Therapeutic agents and methods of use thereof for the treatment of melanoma

ABSTRACT

The present invention provides angiogenesis inhibitor compounds comprising a MetAP-2 inhibitory core coupled to a peptide, as well as pharmaceutical compositions comprising the angiogenesis inhibitor compounds and a pharmaceutically acceptable carrier. The present invention also provides methods of treating an angiogenic disease, e.g., cancer, in a subject by administering to the subject a therapeutically effective amount of one or more of the angiogenesis inhibitor compounds of the invention.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 10/001,945, filed Nov. 1, 2001, now U.S. Pat. No.7,084,108 issued on Aug. 1, 2006; which in turn is acontinuation-in-part of U.S. patent application Ser. No. 09/972,772,filed Oct. 5, 2001, now U.S. Pat. No. 7,037,890, issued May 2, 2006;which in turn is a continuation-in-part of U.S. patent application Ser.No. 09/704,251, filed Nov. 1, 2000, now U.S. Pat. No. 6,548,477, issuedApr. 15, 2003. The entire contents of each of the aforementionedapplications are hereby incorporated in their entirety by reference.

BACKGROUND OF THE INVENTION

Angiogenesis is the fundamental process by which new blood vessels areformed and is essential to a variety of normal body activities (such asreproduction, development and wound repair). Although the process is notcompletely understood, it is believed to involve a complex interplay ofmolecules which both stimulate and inhibit the growth of endothelialcells, the primary cells of the capillary blood vessels. Under normalconditions, these molecules appear to maintain the microvasculature in aquiescent state (i.e., one of no capillary growth) for prolonged periodswhich may last for as long as weeks or in some cases, decades. Whennecessary, however, (such as during wound repair), these same cells canundergo rapid proliferation and turnover within a 5 day period (Folkman,J. and Shing, Y., Journal of Biological Chemistry, 267(16): 10931-10934,and Folkman, J. and Klagsbrun, M. (1987) Science, 235: 442-447).

Although angiogenesis is a highly regulated process under normalconditions, many diseases (characterized as “angiogenic diseases”) aredriven by persistent unregulated angiogenesis. Otherwise stated,unregulated angiogenesis may either cause a particular disease directlyor exacerbate an existing pathological condition. For example, ocularneovacularization has been implicated as the most common cause ofblindness and dominates approximately 20 eye diseases. In certainexisting conditions such as arthritis, newly formed capillary bloodvessels invade the joints and destroy cartilage. In diabetes, newcapillaries formed in the retina invade the vitreous, bleed, and causeblindness. Growth and metastasis of solid tumors are alsoangiogenesis-dependent (Folkman, J. (1986) Cancer Research 46: 467-473and Folkman, J. (1989) Journal of the National Cancer Institute 82:4-6). It has been shown, for example, that tumors which enlarge togreater than 2 mm, must obtain their own blood supply and do so byinducing the growth of new capillary blood vessels. Once these new bloodvessels become embedded in the tumor, they provide a means for tumorcells to enter the circulation and metastasize to distant sites, such asthe liver, lung or bone (Weidner, N., et al. (1991) The New EnglandJournal of Medicine 324(1):1-8).

Fumagillin is a known compound which has been used as an antimicrobialand antiprotozoal. Its physicochemical properties and method ofproduction are well known (U.S. Pat. No. 2,803,586 and Proc. Nat. Acad.Sci. USA (1962) 48:733-735). Fumagillin and certain types of Fumagillinanalogs have also been reported to exhibit anti-angiogenic activity.However, the use of such inhibitors (e.g., TNP-470) may be limited bytheir rapid metabolic degradation, erratic blood levels, and bydose-limiting central nervous system (CNS) side effects.

Accordingly, there is still a need for angiogenesis inhibitors which aremore potent, less neurotoxic, more stable, and/or have longer serumhalf-lives.

SUMMARY OF THE INVENTION

The present invention provides angiogenesis inhibitor compounds whichcomprise a core, e.g., a Fumagillin core, that is believed to inhibitmethionine aminopeptidase 2 (MetAP-2), coupled to a peptide. The presentinvention is based, at least in part, on the discovery that coupling theMetAP-2 inhibitory core to an amino acid residue or an amino acidderivative prevents the metabolic degradation of the angiogenesisinhibitor compound to ensure a superior pharmacokinetic profile andlimits CNS side effects by altering the ability of the angiogenesisinhibitor compound to cross the blood brain barrier. The presentinvention is also based, at least in part, on the discovery thatcoupling the MetAP-2 inhibitory core to a peptide comprising asite-directed sequence allows for a cell specific delivery of theangiogenesis inhibitor compound and limits the toxicity of theangiogenesis inhibitor compound.

Accordingly, the present invention provides compounds of Formula I,

In Formula I, A is a MetAP-2 inhibitory core, W is O or NR₂, and R₁ andR₂ are each, independently, hydrogen or alkyl; X is alkylene orsubstituted alkylene, preferably linear C₁-C₆-alkylene; n is 0 or 1; R₃and R₄ are each, independently, hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted aryl or arylalkyl or substituted orunsubstituted heteroaryl or heteroalkyl. R₃ and R₄ can also, togetherwith the carbon atom to which they are attached, form a carbocyclic orheterocyclic group; or R₁ and R₄ together can form an alkylene group; Zis —C(O)—, alkylene-C(O)— or alkylene; and P is a peptide comprisingfrom 1 to about 100 amino acid residues attached at its amino terminusto Z or a group OR₅ or N(R₆)R₇, wherein R₅, R₆ and R₇ are each,independently, hydrogen, alkyl, substituted alkyl, azacycloalkyl orsubstituted azacycloalkyl. R₆ and R₇ can also form, together with thenitrogen atom to which they are attached, a substituted or unsubstitutedheterocyclic ring structure.

In another embodiment of the compounds of Formula I, W, X, n, R₁, R₃ andR₄ have the meanings given above for these variables; Z is —O—, —NR₈—,alkylene-O— or alkylene-NR₈—, where R₈ is hydrogen or alkyl; and P ishydrogen, alkyl, preferably normal or branched C₁-C₄-alkyl or a peptideconsisting of from 1 to about 100 amino acid residues attached at itscarboxy terminus to Z.

In compounds of Formula I, when any of R₁-R₈ is an alkyl group,preferred alkyl groups are substituted or unsubstituted normal, branchedor cyclic C₁-C₆ alkyl groups. Particularly preferred alkyl groups arenormal or branched C₁-C₄ alkyl groups. A substituted alkyl groupincludes at least one non-hydrogen substituent, such as an amino group,an alkylamino group or a dialkylamino group; a halogen, such as afluoro, chloro, bromo or iodo substituent; or hydroxyl.

When at least one of R₃ and R₄ is a substituted or unsubstituted aryl orheteroaryl group, preferred groups include substituted and unsubstitutedphenyl, naphthyl, indolyl, imidazoly and pyridyl. When at least one ofR₃ and R₄ is substituted or unsubstituted arylalkyl or heteroarylalkyl,preferred groups include substituted and unsubstituted benzyl,naphthylmethyl, indolylmethyl, imidazolylmethyl and pyridylmethylgroups. Preferred substituents on aryl, heteroaryl, arylalkyl andheteroarylalkyl groups are independently selected from the groupconsisting of amino, alkyl-substituted amino, halogens, such as fluoro,chloro, bromo and iodo; hydroxyl groups and alkyl groups, preferablynormal or branched C₁-C₆-alkyl groups, most preferably methyl groups. Xis preferably linear C₁-C₆-alkylene, more preferably C₁-C₄-alkylene andmost preferably methylene or ethylene. When Z is alkylene-C(O)—,alkylene-O— or alkylene-NR₈, the alkylene group is preferably linearC₁-C₆-alkylene, more preferably C₁-C₄-alkylene and most preferablymethylene or ethylene.

R₆ and R₇, in addition to alkyl, substituted alkyl or hydrogen, can eachalso independently be a substituted or unsubstituted azacycloalkyl groupor a substituted or unsubstituted azacycloalkylalkyl group. Suitablesubstituted azacycloalkyl groups include azacycloalkyl groups which havean N-alkyl substituent, preferably an N—C₁-C₄-alkyl substituent and morepreferably an N-methyl substituent. R₆ and R₇ can also, together withthe nitrogen atom to which they are attached, form a heterocyclic ringsystem, such as a substituted or unsubstituted five or six-membered aza-or diazacycloalkyl group. Preferably, the diazacycloalkyl group includesan N-alkyl substituent, such as an N—C₁-C₄-alkyl substituent or, morepreferably, an N-methyl substituent.

In particularly preferred embodiments, —N(R₆)R₇ is NH₂ or one of thegroups shown below:

Preferably, the compounds of Formula I do not include compounds whereinZ is —O—, P is hydrogen, R₃ and R₄ are both hydrogen, n is 1 and X ismethylene. Preferably, the compounds of Formula I further do not includecompounds wherein Z is methylene-O—, R₃ and R₄ are both hydrogen, and nis 0.

In another aspect, the present invention is directed to angiogenesisinhibitor compounds of Formula XV,

where A is a MetAP-2 inhibitory core and W is O or NR. In oneembodiment, Z is —C(O)— or -alkylene-C(O)— and P is NHR, OR or a peptideconsisting of one to about one hundred amino acid residues connected atthe N-terminus to Z. In this embodiment, Q is hydrogen, linear, branchedor cyclic alkyl or aryl, provided that when P is —OR, Q is not hydrogen.

In another embodiment, Z is -alkylene-O— or -alkylene-N(R)— and P ishydrogen or a peptide consisting of from one to about one hundred aminoacid residues connected to Z at the carboxyl terminus. In thisembodiment, Q is hydrogen, linear, branched or cyclic alkyl or aryl,provided that when P is hydrogen, Q is not hydrogen.

In the angiogenesis inhibitor compounds of Formula XV, each R is,independently, hydrogen or alkyl.

In another aspect, the invention features pharmaceutical compositionscomprising the angiogenesis inhibitor compounds of Formula I or XV and apharmaceutically acceptable carrier.

In yet another aspect, the invention features a method of treating anangiogenic disease, e.g., cancer (such as lung cancer, brain cancer,kidney cancer, colon cancer, liver cancer, pancreatic cancer, stomachcancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer,melanoma, and metastatic versions of any of the preceding cancers), in asubject. The method includes administering to the subject atherapeutically effective amount of one or more angiogenesis inhibitorcompounds of Formula I or XV.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the results from the rat aortic ring assay(RARA) used to test the ability of the angiogenesis inhibitor compoundsof the invention to inhibit angiogenesis.

FIG. 2 is a graph depicting the mean daily clinical score for each ofthe four groups of rats tested over the 19 day period followinginduction of clinical arthritis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds useful as angiogenesisinhibitors and methods for using these compounds in the treatment ofangiogenic diseases. Without intending to be limited by theory, it isbelieved that the angiogenesis inhibitor compounds of the inventioninhibit angiogenesis by inhibiting methionine aminopeptidase 2(MetAP-2), an enzyme which cleaves the N-terminal methionine residue ofnewly synthesized proteins to produce the active form of the protein. Atthe same time, the presence of a peptide in the angiogenesis inhibitorcompounds of the invention prevents the metabolic degradation of theangiogenesis inhibitor compounds and ensures a superior pharmacokineticprofile. The presence of the peptide in the angiogenesis inhibitorcompounds of the invention also alters the ability of the angiogenesisinhibitor compound to cross the blood brain barrier to, for example,limit CNS side effects (such as CNS toxicity). The presence of peptidescomprising a site-directed sequence in the angiogenesis inhibitorcompounds of the invention allows for a site-specific delivery of theangiogenesis inhibitor compounds and, thus, limits the toxicity of theangiogenesis inhibitor compounds.

The angiogenesis inhibitor compounds of the invention comprise a MetAP-2inhibitory core and a peptide attached, directly or indirectly, thereto.In one embodiment, the invention provides angiogenesis inhibitorcompounds of Formula I

In Formula I, A is a MetAP-2 inhibitory core, W is O or NR₂, and R₁ andR₂ are each, independently, hydrogen or alkyl; X is alkylene orsubstituted alkylene, preferably linear C₁-C₆-alkylene; n is 0 or 1; R₃and R₄ are each, independently, hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted aryl or arylalkyl or substituted orunsubstituted heteroaryl or heteroalkyl. R₃ and R₄ can also, togetherwith the carbon atom to which they are attached, form a carbocyclic orheterocyclic group; or R₁ and R₄ together can form an alkylene group; Zis —C(O)—, alkylene-C(O)— or alkylene; and P is a peptide comprisingfrom 1 to about 100 amino acid residues attached at its amino terminusto Z or a group OR₅ or N(R₆)R₇, wherein R₅, R₆ and R₇ are each,independently, hydrogen, alkyl, substituted alkyl, azacycloalkyl orsubstituted azacycloalkyl. R₆ and R₇ can also form, together with thenitrogen atom to which they are attached, a substituted or unsubstitutedheterocyclic ring structure.

In another embodiment of the compounds of Formula I, W, X, n, R₁, R₃ andR₄ have the meanings given above for these variables; Z is —O—, —NR₈—,alkylene-O— or alkylene-NR₈—, where R₈ is hydrogen or alkyl; and P ishydrogen, alkyl, preferably normal or branched C₁-C₄-alkyl or a peptideconsisting of from 1 to about 100 amino acid residues attached at itscarboxy terminus to Z.

In compounds of Formula I, when any of R₁-R₈ is an alkyl group,preferred alkyl groups are substituted or unsubstituted normal, branchedor cyclic C₁-C₆ alkyl groups. Particularly preferred alkyl groups arenormal or branched C₁-C₄ alkyl groups. A substituted alkyl groupincludes at least one non-hydrogen substituent, such as an amino group,an alkylamino group or a dialkylamino group; a halogen, such as afluoro, chloro, bromo or iodo substituent; or hydroxyl.

When at least one of R₃ and R₄ is a substituted or unsubstituted aryl orheteroaryl group, preferred groups include substituted and unsubstitutedphenyl, naphthyl, indolyl, imidazolyl and pyridyl. When at least one ofR₃ and R₄ is substituted or unsubstituted arylalkyl or heteroarylalkyl,preferred groups include substituted and unsubstituted benzyl,naphthylmethyl, indolylmethyl, imidazolylmethyl and pyridylmethylgroups. Preferred substituents on aryl, heteroaryl, arylalkyl andheteroarylalkyl groups are independently selected from the groupconsisting of amino, alkyl-substituted amino, halogens, such as fluoro,chloro, bromo and iodo; hydroxyl groups and alkyl groups, preferablynormal or branched C₁-C₆-alkyl groups, most preferably methyl groups. Xis preferably linear C₁-C₆-alkylene, more preferably C₁-C₄-alkylene andmost preferably methylene or ethylene. When Z is alkylene-C(O)—,alkylene-O— or alkylene-NR₈, the alkylene group is preferably linearC₁-C₆-alkylene, more preferably C₁-C₄-alkylene and most preferablymethylene or ethylene.

R₆ and R₇, in addition to alkyl, substituted alkyl or hydrogen, can eachalso independently be a substituted or unsubstituted azacycloalkyl groupor a substituted or unsubstituted azacycloalkylalkyl group. Suitablesubstituted azacycloalkyl groups include azacycloalkyl groups which havean N-alkyl substituent, preferably an N-C₁-C₄-alkyl substituent and morepreferably an N-methyl substituent. R₆ and R₇ can also, together withthe nitrogen atom to which they are attached, form a heterocyclic ringsystem, such as a substituted or unsubstituted five or six-membered aza-or diazacycloalkyl group. Preferably, the diazacycloalkyl group includesan N-alkyl substituent, such as an N—C₁-C₄-alkyl substituent or, morepreferably, an N-methyl substituent.

In particularly preferred embodiments, —N(R₆)R₇ is NH₂ or one of thegroups shown below:

Preferably, the compounds of Formula I do not include compounds whereinZ is —O—, P is hydrogen, R₃ and R₄ are both hydrogen, n is 1 and X ismethylene. Preferably, the compounds of Formula I further do not includecompounds wherein Z is methylene-O—, R₃ and R₄ are both hydrogen, and nis 0.

In another embodiment, the invention provides angiogenesis inhibitorcompounds of Formula XV,

where A is a MetAP-2 inhibitory core and W is O or NR. In oneembodiment, Z is —C(O)— or -alkylene-C(O)— and P is NHR, OR or a peptideconsisting of one to about one hundred amino acid residues connected atthe N-terminus to Z. In this embodiment, Q is hydrogen, linear, branchedor cyclic alkyl or aryl, provided that when P is —OR, Q is not hydrogen.Z is preferably —C(O)— or C₁-C₄-alkylene-C(O)—, and, more preferably,—C(O)— or C₁-C₂-alkylene-C(O)—. Q is preferably linear, branched orcyclic C₁-C₆-alkyl, phenyl or naphthyl. More preferably, Q is isopropyl,phenyl or cyclohexyl.

In another embodiment, Z is -alkylene-O— or -alkylene-N(R)—, wherealkylene is, preferably, C₁-C₆-alkylene, more preferably C₁-C₄-alkyleneand, most preferably, C₁-C₂-alkylene. P is hydrogen or a peptideconsisting of from one to about one hundred amino acid residuesconnected to Z at the carboxyl terminus. In this embodiment, Q ishydrogen, linear, branched or cyclic alkyl or aryl, provided that when Pis hydrogen, Q is not hydrogen. Q is preferably linear, branched orcyclic C₁-C₆-alkyl , phenyl or naphthyl. More preferably, Q isisopropyl, phenyl or cyclohexyl.

In the compounds of Formula XV, each R is, independently, hydrogen oralkyl. In one embodiment, each R is, independently, hydrogen or linear,branched or cyclic C₁-C₆-alkyl. Preferably, each R is, independently,hydrogen or linear or branched C₁-C₄-alkyl. More preferably, each R is,independently, hydrogen or methyl. In the most preferred embodiments,each R is hydrogen.

In Formulas I and XV, A is a MetAP-2 inhibitory core. As used herein, a“MetAP-2 inhibitory core” includes a moiety able to inhibit the activityof methionine aminopeptidase 2 (MetAP-2), e.g., the ability of MetAP-2to cleave the N-terminal methionine residue of newly synthesizedproteins to produce the active form of the protein. Preferred MetAP-2inhibitory cores are Fumagillin derived structures.

Suitable MetAP-2 inhibitory cores include the cores of Formula II,

where R¹ is hydrogen or alkoxy, preferably C₁-C₄-alkoxy and morepreferably, methoxy. R² is hydrogen or hydroxy; and R³ is hydrogen oralkyl, preferably C₁-C₄-alkyl and more preferably, hydrogen. D is linearor branched alkyl, preferably C₁-C₆-alkyl; arylalkyl, preferablyaryl-C₁-C₄-alkyl and more preferably phenyl-C₁-C₄-alkyl; or D is of thestructure

where the dashed line represents a single bond or a double bond.

A can also be a MetAP-2 inhibitory core of Formula III,

Where R¹, R², R³ and D have the meanings given above for Formula II, andX is a leaving group, such as a halogen.

Examples of suitable MetAP-2 inhibitory cores include, but are notlimited to, the following.

In each of Formulas IV-X, the indicated valence on the ring carbon isthe point of attachment of the structural variable W, as set forth inFormulas I-XV. In Formula IX, p is an integer from 0 to 10, preferably1-4. In Formulas IV, V and VI-IX, R₁ is hydrogen or C₁-C₄-alkoxy,preferably methoxy. In Formulas IV and V, the dashed line indicates thatthe bond can be a double bond or a single bond. In Formula V, Xrepresents a leaving group, such as a thioalkoxy group, a thioaryloxygroup, a halogen or a dialkylsulfinium group. In Formulas IV and V, R₂is H, OH, amino, C₁-C₄-alkylamino or di(C₁-C₄-alkyl)amino), preferablyH. In formulas in which the stereochemistry of a particular stereocenteris not indicated, that stereocenter can have either of the possiblestereochemistries, consistent with the ability of the angiogenesisinhibitor compound to inhibit the activity of MetAP-2.

In particularly preferred embodiments, A is the MetAP-2 inhibitory coreof Formula X below.

As used herein, the terms “P” and “peptide” include compounds comprisingfrom 1 to about 100 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acidresidues). In preferred embodiments, the peptide includes compoundscomprising less than about 90, 80, 70, 60, 50, 40, 30, 20, or 10 aminoacid residues, preferably about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60,1-70, 1-80, or 1-90 amino acid residues. The peptides may be natural orsynthetically made. The amino acid residues are preferably α-amino acidresidues. For example, the amino acid residues can be independentlyselected from among the twenty naturally occurring amino acid residues,the D-enantiomers of the twenty natural amino acid residues, and mayalso be non-natural amino acid residues (e.g., norleucine, norvaline,phenylglycine, β-alanine, or a peptide mimetic such as3-amino-methylbenzoic acid). In one embodiment, the amino acid residuesare independently selected from residues of Formula XI, Formula XII, andFormula XIII.

In Formula XI, X₁ is hydrogen, a side chain of one of the twentynaturally-occurring amino acid residues, a linear, branched or cyclicC₁-C₈-alkyl group, an aryl group, such as a phenyl or naphthyl group, anaryl-C₁-C₄-alkyl group, a heteroaryl group, such as a pyridyl, thienyl,pyrrolyl, or furyl group, or a heteroaryl-C₁-C₄-alkyl group; and X₂ ishydrogen a linear, branched or cyclic C₁-C₈-alkyl group, an aryl group,such as a phenyl or naphthyl group, an aryl-C₁-C₄-alkyl group or aheteroaryl group as described above for X₁. Preferably, X₂ is hydrogen.In Formula XII, Y is methylene, oxygen, sulfur or NH, and a and b areeach, independently, 0-4, provided that the sum of a and b is between 1and 4. Formulas XI and XII encompass α-amino acid residues having eithera D or an L stereochemistry at the alpha carbon atom. One or more of theamino acid residues can also be an amino acid residue other than anα-amino acid residue, such as a β-, γ- or ε-amino acid residue. Suitableexamples of such amino acid residues are of Formula XIII, wherein q isan integer of from 2 to about 6, and each X₁ and X₂ independently havethe meanings given above for these variables in Formula XI.

In a preferred embodiment, the peptide used in the angiogenesisinhibitor compounds of the invention may include a site-directedsequence in order to increase the specificity of binding of theangiogenesis inhibitor compound to a cell surface of interest. As usedherein, the term “site-directed sequence” is intended to include anyamino acid sequence (e.g., comprised of natural or non natural aminoacid residues) which serves to limit exposure of the angiogenesisinhibitor compound to the periphery and/or which serves to direct theangiogenesis inhibitor compound to a site of interest, e.g., a site ofangiogenesis or aberrant cellular proliferation.

The peptide contained within the angiogenesis inhibitor compounds of theinvention may include a peptide cleavage site for an enzyme which isexpressed at sites of angiogenesis or aberrant cell proliferation,allowing tissue-selective delivery of a cell-permeable activeangiogenesis inhibitor compound or fragment thereof (e.g., a fragmentcontaining the MetAP-2 inhibitory core of the angiogenesis inhibitorcompound). The peptide may also include a sequence which is a ligand fora cell surface receptor which is expressed at a site of angiogenesis oraberrant cell proliferation, thereby targeting angiogenesis inhibitorcompounds to a cell surface of interest. For example, a peptidecontained within the angiogenesis inhibitor compounds of the inventioncan include a cleavage site for a matrix metalloproteinase, or anintegrin binding RGD (Arg-Gly-Asp) sequence, or a combination of both anenzyme “cleavage” sequence and a cell surface “ligand” which serve totarget the angiogenesis inhibitor compound to the membrane of anendothelial cell. However, the selection of a peptide sequence must besuch that the active angiogenesis inhibitor compound is available to bedelivered to the cells in which MetAP-2 inhibition is desired.

For example, a sequence that is cleaved by a matrix matalloproteinaseproduces a product that contains the MetAP-2 inhibitory core, a couplinggroup, and a peptide fragment. Sequences are selected so that the activeangiogenesis inhibitor compound, e.g., the active angiogenesis inhibitorcompound generated by the matrix matalloproteinase cleavage, is cellpermeable. Preferably, the active angiogenesis inhibitor compound doesnot contain a free acid after the cleavage.

In one embodiment, the peptide includes a cleavage site for a matrixmetalloprotease, such as matrix metalloprotease-2 (MMP-2), MMP-1, MMP-3,MMP-7, MMP-8, MMP-9, MMP-12, MMP-13 or MMP-26. Preferably, the peptideincludes a cleavage site for MMP-2 or MMP-9. For example, the peptidecan comprise the sequence -Pro-Leu-Gly-Xaa- (SEQ ID NO:1), where Xaa isany naturally occurring amino acid residue consistent with matrixmetalloprotease (MMP) cleavage at the Gly-Xaa bond. Xaa is preferably ahydrophobic amino acid residue, such as tryptophan, phenylalanine,methionine, leucine, isoleucine, proline, and valine.

Other suitable sequences include sequences comprising one or more ofPro-Cha-Gly-Cys(Me)-His (SEQ ID NO:2); Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg(SEQ ID NO:3); Pro-Gln-Gly-Ile-Ala-Gly-Trp (SEQ ID NO:4);Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg (SEQ ID NO:5);Pro-Leu-Gly-Met-Trp-Ser-Arg (SEQ ID NO:35);Pro-Leu-Gly-Leu-Trp-Ala-D-Arg (SEQ ID NO:6); Pro-Leu-Ala-Leu-Trp-Ala-Arg(SEQ ID NO:7); Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:8);Pro-Leu-Ala-Tyr-Trp-Ala-Arg (SEQ ID NO:9); Pro-Tyr-Ala-Tyr-Trp-Met-Arg(SEQ ID NO:10); Pro-Cha-Gly-Nva-His-Ala (SEQ ID NO:11); Pro-Leu-Ala-Nva(SEQ ID NO:12); Pro-Leu-Gly-Leu (SEQ ID NO:13); Pro-Leu-Gly-Ala (SEQ IDNO:14); Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (SEQ ID NO:15);Pro-Cha-Ala-Abu-Cys(Me)-His-Ala (SEQ ID NO:16);Pro-Cha-Ala-Gly-Cys(Me)-His-Ala (SEQ ID NO:17);Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu (SEQ ID NO:18);Pro-Lys-Pro-Leu-Ala-Leu (SEQ ID NO:19);Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met (SEQ ID NO:20);Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:21);Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:22); andArg-Pro-Lys-Pro-Leu-Ala-Nva-Trp (SEQ ID NO:23). These sequences identifythe natural amino acid residues using the customary three-letterabbreviations; the following abbreviations represent the indicatednon-natural amino acids: Abu=L-a-aminobutyryl; Cha=L-cyclohexylalanine;Nva=L-norvaline.

In certain embodiments, P is an amino acid sequence selected from thegroup consisting of Ac-Pro-Leu-Gly-Met-Trp-Ala (SEQ ID NO:24);Gly-Pro-Leu-Gly-Met-His-Ala-Gly (SEQ ID NO:25); Gly-Pro-Leu-(Me)Gly (SEQID NO:26); Gly-Pro-Leu-Gly (SEQ ID NO:27); Gly-Met-Gly-Leu-Pro (SEQ IDNO:28); Ala-Met-Gly-Ile-Pro (SEQ ID NO:29);Gly-Arg-Gly-Asp-(O-Me-Tyr)-Arg-Glu (SEQ ID NO:30);Gly-Arg-Gly-Asp-Ser-Pro (SEQ ID NO:31); Gly-Arg-Gly-Asp (SEQ ID NO:32);Asp-Gly-Arg; Ac-Pro-Leu-Gly-Met-Ala (SEQ ID NO:34);Ac-Arg-Gly-Asp-Ser-Pro-Leu-Gly-Met-Trp-Ala (SEQ ID NO:33);Ac-Pro-Leu-Gly-Met-Gly (SEQ ID NO:36); Met-Trp-Ala (SEQ ID NO:37);Met-Gly (SEQ ID NO:38); Gly-Pro-Leu-Gly-Met-Trp-Ala-Gly (SEQ ID NO:39);and Gly-Arg-Gly-(3-amino-3-pyridylpropionic acid) (SEQ ID NO:40). (Ac inthe foregoing sequences represents an Acetyl group).

The peptide can be attached to the MetAP-2 inhibitory core at either itsN-terminus or C-terminus. When the peptide is attached to the MetAP-2inhibitory core at its C-terminus, the N-terminus of the peptide can be—NR₂R₃, where R₂ is hydrogen, alkyl or arylalkyl and R₃ is hydrogen,alkyl, arylalkyl or acyl. When the peptide is attached to the MetAP-2inhibitory core at its N-terminus, the C-terminus can be —C(O)R₄, whereR₄ is —OH, —O-alkyl, —O-arylalkyl, or —NR₂R₃, where R₂ is hydrogen,alkyl or arylalkyl and R₃ is hydrogen, alkyl, arylalkyl or acyl. In thisembodiment, the C-terminal residue can also be present in a reducedform, such as the corresponding primary alcohol.

The present invention also includes pharmaceutically acceptable salts ofthe angiogenesis inhibitor compounds of the invention. A“pharmaceutically acceptable salt” includes a salt that retains thedesired biological activity of the parent angiogenesis inhibitorcompound and does not impart any undesired toxicological effects.Examples of such salts are salts of acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, phosporic acid, nitric acid, and thelike; acetic acid, oxalic acid, tartaric acid, succinic acid, malicacid, benzoic acid, pamoic acid, alginic acid, methanesulfonic acid,naphthalenesulfonic acid, and the like. Also included are salts ofcations such as sodium, potassium, lithium, zinc, copper, barium,bismuth, calcium, and the like; or organic cations such astrialkylammonium. Combinations of the above salts are also useful.

Preferred Angiogenesis Inhibitor Compounds of Formula I

One set of particularly preferred angiogenesis inhibitor compounds ofthe invention includes compounds in which A is the MetAP-2 inhibitorycore of Formula X, W is O or NR₂, and the structure

is represented by the structures set forth below.

Preferred Angiogenesis Inhibitor Compounds of Formula XV

A preferred subset of the angiogenesis inhibitor compounds of Formula XVcomprises Formula XIV shown below.

In one embodiment, W is O or NR. Z is —C(O) or -alkylene-C(O)—,preferably C1-C4-alkylene-C(O)—. R is hydrogen or a C₁-C₄-alkyl. Q ishydrogen; linear, branched or cyclic C₁-C₆-alkyl; or aryl. R₁is hydroxy,C₁-C₄-alkoxy or halogen. P is NH₂, OR or a peptide attached to Z at itsN-terminus and comprising from 1 to 100 amino acid residuesindependently selected from naturally occurring amino acid residues,D-enantiomers of the naturally occurring amino acid residues andnon-natural amino acid residues. When Q is H, P is not NH2 or OR. Inpreferred embodiments, W is O or NH; Q is isopropyl; R₁ is methoxy; Pcomprises from 1 to 15 amino acid residues; and the dashed line presentin Formula XIV represents a double bond. In particularly preferredembodiments, W is O, and P comprises 10 or fewer amino acid residues.

In another embodiment of the compounds of Formula XIV, W is O or NR. Zis alkylene-O or alkylene-NR, preferably C1-C4-alkylene-O orC1-C4-alkylene-NR—. R is hydrogen or a C₁-C₄-alkyl. Q is hydrogen;linear, branched or cyclic C₁-C₆-alkyl; or aryl. R₁ is hydroxy,C₁-C₄-alkoxy or halogen. P is hydrogen or a peptide attached to Z at itsC-terminus and comprising from 1 to 100 amino acid residuesindependently selected from naturally occurring amino acid residues,D-enantiomers of the naturally occurring amino acid residues andnon-natural amino acid residues. When Q is H, P is not H. In preferredembodiments, W is O or NH; Q is isopropyl; R₁ is methoxy; P comprisesfrom 1 to 15 amino acid residues; and the dashed line present in FormulaXIV represents a double bond. In particularly preferred embodiments, Wis O, and P comprises 10 or fewer amino acid residues or P is hydrogen.

One set of particularly preferred angiogenesis inhibitor compounds ofthe invention is represented by the structures set forth below.

Methods of Using the Angiogenesis Inhibitor Compounds for the Treatmentof Angiogenic Disease

In another embodiment, the present invention provides a method oftreating an angiogenic disease in a subject. The method includesadministering to the subject a therapeutically effective amount of anangiogenesis inhibitor compound of the present invention, therebytreating the angiogenic disease in the subject.

As used herein, the term “angiogenic disease” includes a disease,disorder, or condition characterized or caused by aberrant or unwanted,e.g., stimulated or suppressed, formation of blood vessels(angiogenesis). Aberrant or unwanted angiogenesis may either cause aparticular disease directly or exacerbate an existing pathologicalcondition. Examples of angiogenic diseases include ocular disorders,e.g., diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, retrolental fibroplasia, neovascular glaucoma, rubeosis,retinal neovascularization due to macular degeneration, hypoxia,angiogenesis in the eye associated with infection or surgicalintervention, ocular tumors and trachoma, and other abnormalneovascularization conditions of the eye, where neovascularization maylead to blindness; disorders affecting the skin, e.g., psoriasis andpyogenic granuloma; cancer, e.g., carcinomas and sarcomas, whereprogressive growth is dependent upon the continuous induction ofangiogenesis by these tumor cells, lung cancer, brain cancer, kidneycancer, colon cancer, liver cancer, pancreatic cancer, stomach cancer,prostate cancer, breast cancer, ovarian cancer, cervical cancer,melanoma, and metastatic versions of any of the preceding cancers;pediatric disorders, e.g., angiofibroma, and hemophiliac joints; bloodvessel diseases such as hemangiomas, and capillary proliferation withinatherosclerotic plaques; disorders associated with surgery, e.g.,hypertrophic scars, wound granulation and vascular adhesions; andautoimmune diseases such as rheumatoid, immune and degenerativearthritis, where new vessels in the joint may destroy articularcartilage and scleroderma.

The term angiogenic disease also includes diseases characterized byexcessive or abnormal stimulation of endothelial cells, including butnot limited to intestinal adhesions, Crohn's disease, atherosclerosis,scleroderma, and hypertrophic scars, i.e., keloids; diseases that haveangiogenesis as a pathologic consequence such as cat scratch disease(Rochele ninalia quintosa) and ulcers (Helicobacter pylori). Inaddition, the angiogenesis inhibitor compounds of the present inventionare useful as birth control agents (by virtue of their ability toinhibit the angiogenesis dependent ovulation and establishment of theplacenta) and may also be used to reduce bleeding by administration to asubject prior to surgery.

As used herein, the term “subject” includes warm-blooded animals,preferably mammals, including humans. In a preferred embodiment, thesubject is a primate. In an even more preferred embodiment, the primateis a human.

As used herein, the term “administering” to a subject includesdispensing, delivering or applying an angiogenesis inhibitor compound,e.g., an angiogenesis inhibitor compound in a pharmaceutical formulation(as described herein), to a subject by any suitable route for deliveryof the compound to the desired location in the subject, includingdelivery by either the parenteral or oral route, intramuscularinjection, subcutaneous/intradermal injection, intravenous injection,buccal administration, transdermal delivery and administration by therectal, colonic, vaginal, intranasal or respiratory tract route.

As used herein, the term “effective amount” includes an amounteffective, at dosages and for periods of time necessary, to achieve thedesired result, e.g., sufficient to treat an angiogenic disease in asubject. An effective amount of an angiogenesis inhibitor compound, asdefined herein may vary according to factors such as the disease state,age, and weight of the subject, and the ability of the angiogenesisinhibitor compound to elicit a desired response in the subject. Dosageregimens may be adjusted to provide the optimum therapeutic response. Aneffective amount is also one in which any toxic or detrimental effects(e.g., side effects) of the angiogenesis inhibitor compound areoutweighed by the therapeutically beneficial effects.

A therapeutically effective amount of an angiogenesis inhibitor compound(i.e., an effective dosage) may range from about 0.001 to 30 mg/kg bodyweight, preferably about 0.01 to 25 mg/kg body weight, more preferablyabout 0.1 to 20 mg/kg body weight, and even more preferably about 1 to10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg bodyweight. The skilled artisan will appreciate that certain factors mayinfluence the dosage required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of an angiogenesis inhibitor compoundcan include a single treatment or, preferably, can include a series oftreatments. In one example, a subject is treated with an angiogenesisinhibitor compound in the range of between about 0.1 to 20 mg/kg bodyweight, one time per week for between about 1 to 10 weeks, preferablybetween 2 to 8 weeks, more preferably between about 3 to 7 weeks, andeven more preferably for about 4, 5, or 6 weeks. It will also beappreciated that the effective dosage of an angiogenesis inhibitorcompound used for treatment may increase or decrease over the course ofa particular treatment.

The methods of the invention further include administering to a subjecta therapeutically effective amount of an angiogenesis inhibitor compoundin combination with another pharmaceutically active compound known totreat an angiogenic disease, e.g., a chemotherapeutic agent such asTaxol, Paclitaxel, or Actinomycin D, or an antidiabetic agent such asTolbutamide; or a compound that may potentiate the angiogenesisinhibitory activity of the angiogenesis inhibitor compound, such asheparin or a sulfated cyclodextrin. Other pharmaceutically activecompounds that may be used can be found in Harrison's Principles ofInternal Medicine, Thirteenth Edition, Eds. T. R. Harrison et al.McGraw-Hill N.Y., N.Y.; and the Physicians Desk Reference 50th Edition1997, Oradell N.J., Medical Economics Co., the complete contents ofwhich are expressly incorporated herein by reference. The angiogenesisinhibitor compound and the pharmaceutically active compound may beadministered to the subject in the same pharmaceutical composition or indifferent pharmaceutical compositions (at the same time or at differenttimes).

Pharmaceutical Compositions of the Angiogenesis Inhibitor Compounds

The present invention also provides pharmaceutically acceptableformulations comprising one or more angiogenesis inhibitor compounds.Such pharmaceutically acceptable formulations typically include one ormore angiogenesis inhibitor compounds as well as a pharmaceuticallyacceptable carrier(s) and/or excipient(s). As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and anti fungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theangiogenesis inhibitor compounds, use thereof in the pharmaceuticalcompositions is contemplated.

Supplementary pharmaceutically active compounds known to treat anangiogenic disease, e.g., a chemotherapeutic agent such as Taxol,Paclitaxel, or Actinomycin D, or an antidiabetic agent such asTolbutamide; or compounds that may potentiate the angiogenesisinhibitory activity of the angiogenesis inhibitor compound, such asheparin or a sulfated cyclodextrin, can also be incorporated into thecompositions of the invention. Suitable pharmaceutically activecompounds that may be used can be found in Harrison's Principles ofInternal Medicine (supra).

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the pharmaceutical composition must be sterile and should befluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theangiogenesis inhibitor compound in the required amount in an appropriatesolvent with one or a combination of the ingredients enumerated above,as required, followed by filtered sterilization. Generally, dispersionsare prepared by incorporating the angiogenesis inhibitor compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the angiogenesis inhibitor compound plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, theangiogenesis inhibitor compound can be incorporated with excipients andused in the form of tablets, troches, or capsules. Oral compositions canalso include an enteric coating. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the angiogenesisinhibitor compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the angiogenesis inhibitor compoundsare delivered in the form of an aerosol spray from pressured containeror dispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the angiogenesis inhibitor compounds areformulated into ointments, salves, gels, or creams as generally known inthe art.

The angiogenesis inhibitor compounds can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the angiogenesis inhibitor compounds are preparedwith carriers that will protect the compound against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811, U.S. Pat. No. 5,455,044 and U.S. Pat. No. 5,576,018, and U.S.Pat. No. 4,883,666, the contents of all of which are incorporated hereinby reference.

The angiogenesis inhibitor compounds of the invention can also beincorporated into pharmaceutical compositions which allow for thesustained delivery of the angiogenesis inhibitor compounds to a subjectfor a period of at least several weeks to a month or more. Suchformulations are described in U.S. Pat. No. 5,968,895, the contents ofwhich are incorporated herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity ofangiogenesis inhibitor compounds calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms of the inventionare dictated by and directly dependent on the unique characteristics ofthe angiogenesis inhibitor compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such angiogenesis inhibitor compounds for the treatment ofindividuals.

Toxicity and therapeutic efficacy of such angiogenesis inhibitorcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Angiogenesis inhibitorcompounds which exhibit large therapeutic indices are preferred. Whileangiogenesis inhibitor compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchangiogenesis inhibitor compounds to the site of affected tissue in orderto minimize potential damage to uninfected cells and, thereby, reduceside effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch angiogenesis inhibitor compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyangiogenesis inhibitor compounds used in the methods of the invention,the therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the angiogenesis inhibitor compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Assays for Detecting the Activity of the Angiogenesis InhibitorCompounds

The angiogenesis inhibitor compounds of the invention may be tested fortheir ability to modulate (e.g., inhibit or stimulate) angiogenesis in avariety of well known assays, e.g., the rat aortic ring angiogenesisinhibition assay (described herein in Example 27) or in achorioallantoic membrane (CAM) assay.

The CAM assay may be performed essentially as described in Liekens S. etal. (1997) Oncology Research 9: 173-181, the contents of which areincorporated herein by reference. Briefly, fresh fertilized eggs areincubated for 3 days at 37° C. On the third day, the shell is crackedand the egg is placed into a tissue culture plate and incubated at 38°C. For the assay, the angiogenesis inhibitor compound to be tested isattached on a matrix of collagen on a nylon mesh. The mesh is then usedto cover the chorioallantoic membrane and the eggs are incubated at 37°C. If angiogenesis occurs, new capillaries form and grow through themesh within 24 hours. The ability of the angiogenesis inhibitor compound(at various concentrations) to modulate, e.g., inhibit, angiogenesis,e.g., FGF-induced angiogenesis, may then be determined.

The angiogenesis inhibitor compounds of the invention may also be testedfor their ability to modulate (e.g., inhibit or stimulate) humanendothelial cell growth. Human umbilical vein endothelial cells (HUVE)may be isolated by perfusion of an umbilical vein with atrypsin-containing medium. HUVE may then be cultured in GIT medium(Diago Eiyou Kagaku, Co., Japan) supplemented with 2.5% fetal bovineserum and 2.0 ng/ml of recombinant human basic fibroblast growth factor(rbFGF, Biotechnology Research Laboratories, Takeda, Osaka, Japan) at37° C. under 5% CO₂ and 7% O₂. HUVE are then plated on 96-wellmicrotiter plates (Nunc, 1-67008) at a cell density of 2×10³/100 μl ofmedium. The following day, 100 μl of medium containing rbFGF (2 ng/ml atthe final concentration) and each angiogenesis inhibitor compound atvarious concentrations may be added to each well. The angiogenesisinhibitor compounds are dissolved in dimethylsulfoxide (DMSO) and thendiluted with culture medium so that the final DMSO concentration doesnot exceed 0.25% . After a 5-day culture, medium is removed, 100 μl of 1mg/ml of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazoliumbromide) solution is added to the wells, and microtiters are kept at 37°C. for 4 hours. Then, 100 μl of 10% sodium dodecyl sulfate (SDS)solution is added to wells, and the microtiters are kept at 37° C. for5-6 hours. To determine the effects of the angiogenesis inhibitorcompound on cell number, the optical density (590 μm) of each well ismeasured using an optical densitometer.

The ability of the angiogenesis inhibitor compounds of the invention tomodulate capillary endothelial cell migration in vitro may also betested using the Boyden chamber assay (as described in Falk et al.(1980) J. Immunol. Meth. 33:239-247, the contents of which areincorporated herein by reference). Briefly, bovine capillary endothelialcells are plated at 1.5×10⁴ cells per well in serum-free DMEM(Dulbecco's Modified Eagle's Medium) on one side of nucleopore filterspre-coated with fibronectin (7.3 μg fibronectin/ml PBS). An angiogenesisinhibitor compound is dissolved in ethanol and diluted in DMEM so thatthe final concentration of ethanol does not exceed 0.01% . Cells areexposed to endothelial mitogen (Biomedical Technologies, Mass.) at 200μg/ml and different concentrations of the angiogenesis inhibitorcompound in serum-free DMEM for 4 hours at 37° C. At the end of thisincubation, the number of cells that migrate through 8μ pores in thefilters is determined by counting cells with an ocular grid at 100× inquadruplicate.

The ability of the angiogenesis inhibitor compounds of the invention tomodulate tumor growth may be tested in vivo. An animal model, e.g., aC57BL/6N mouse with a mouse reticulum cell sarcoma (M 5076)intraperitoneally transplantated therein, may be used. The tumor cellsin ascites can be collected by centrifugation, and suspended in saline.The cell suspension (2×10⁶ cells/100 μl/mouse) is inoculated into theright flanks of mice. Tumor-bearing mice are then subcutaneously treatedwith the angiogenesis inhibitor compound (at various concentrationssuspended in 5% arabic gum solution containing 1% of ethanol) for 12days beginning one day after the tumor inoculation. The tumor growth maybe determined by measuring tumor size in two directions with calipers atintervals of a few days.

Finally, the ability of the angiogenesis inhibitor compounds of theinvention to modulate the activity of MetAP2 may be tested as follows.Recombinant human MetAP2 may be expressed and purified from insect cellsas described in Li and Chang, (1996) Biochem. Biophys. Res. Commun.227:152-159. Various amounts of angiogenesis inhibitor compound is thenadded to buffer H (10 mM Hepes, pH 7.35, 100 mM KC1, 10% glycerol, and0.1 M Co²⁺) containing 1 nM purified recombinant human MetAP2 andincubated at 37° C. for 30 minutes. To start the enzymatic reaction apeptide containing a methionine residue, e.g., Met-Gly-Met, is added tothe reaction mixture (to a concentration of 1 mM). Released methionineis subsequently quantified at different time points (e.g., at 0, 2, 3,and 5 minutes) using the method of Zou et al. (1995) Mol. Gen Genetics246:247-253).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, are herebyincorporated by reference.

EXAMPLES

Synthetic Methods

Compounds of the invention can be prepared using one or more of thefollowing general methods.

General Procedure A: To a mixture of carbonic acid-(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester 4-nitro-phenyl ester¹ (1, 0.47 mmol; Han, C. K.; Ahn, S. K.; Choi,N. S.; Hong, R. K.; Moon, S. K.; Chun, H. S.; Lee, S. J.; Kim, J. W.;Hong, C. I.; Kim, D.; Yoon, J. H.; No, K. T. Biorg. Med. Chem. Lett.2000, 10, 39-43) and amine (2.35 mmol) in EtOH (9 mL) was addeddropwise, diisopropyl ethyl amine (2.35 mmol). After 3-18 hours, theethanol was removed in vacuo and the crude material was dissolved intoEtOAc (10 mL) and washed with H₂O (2×5 mL), and then brine (5 mL). Theorganic phase was dried over Na₂SO₄ and the solvent removed in vacuo.Purification via flash chromatography (2-5% MeOH/CH₂Cl₂) affordedproduct.General Procedure B, Part I: A solution of (3R, 4S, 5S,6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino)-aceticacid² (2, 0.11 mmol; U.S. Pat. No. 6,017,954) in DMF (1 mL) was added toa 10 mL round bottomed flask containing swelled PS-DCC (0.28 mmol). In aseparate vessel, the peptide (0.04 mmol) was dissolved into DMF (0.5 mL)and neutralized with NMM (0.04 mmol). After 1 hour, the solution ofpeptide was added to the pre-activated acid, and the reaction wascontinued for 5-18 hours. The resin was removed by filtration, washedwith DMF (0.5 mL) and the solvent removed in vacuo. Purification viaHPLC (CH₃CN/H₂O) afforded the product.General Procedure B, Part II: A solution of the product in Part 1 (0.009mmol) was dissolved into MeOH (1 mL) and was treated with Pd/C (2 mg),then subjected to a H₂ atmosphere (38 psi) for 24 hours. The mixture wasthen filtered through Celite, washed with MeOH (0.5 mL) and the solventremoved in vacuo. Purification via HPLC (CH₃CN/H₂O) afforded the productas a white solid.General Procedure C: (1-Hydroxymethyl-methyl-propyl)-carbamic acid (3R,4S, 5S, 6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester (Example 7, 189 mg, 0.46 mmol), acid (0.46 mmol) and DMAP (0.69mmol) were dissolved into anhydrous CH₂Cl₂ (5 mL) and treated withdiisopropylcarbodiimide (0.46 mmol). After 7-18 hours, the solvent wasremoved in vacuo and purification via flash chromatography (MeOH/CH₂Cl₂)afforded the product.

Example 1 2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3-methyl-butyricacid methyl ester

General procedure A was followed using 1 (31 mg, 0.07 mmol), L-valinemethyl ester hydrochloride (58 mg, 0.35 mmol), and DIEA (60 μL, 0.35mmol) in EtOH (2 mL). Purification via flash chromatography (1%MeOH/CH₂Cl₂) afforded the product as a clear oil (10 mg, 0.02 mmol, 33%yield); R_(f)=0.60 (20% EtOAc/CH₂Cl₂); LRMS (m/z) [M+1]⁺440.3(calculated for C₂₃H₃₈NO₇, 440.3).

Example 2 2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3-methyl-butyricacid methyl ester

General procedure A was followed using 1 (41 mg, 0.09 mmol) and D-valinemethyl ester hydrochloride (77 mg, 0.45 mmol), and DIEA (80 μL, 0.45mmol) in EtOH (2 mL). Purification via flash chromatography (1%MeOH/CH₂Cl₂) afforded the product as a clear oil (18 mg, 0.04 mmol, 45%yield); R_(f)=0.39 (20% EtOAc/CH₂Cl₂; LRMS (m/z) [M+1]⁺440.3 (calculatedfor C₂₃H₃₈NO₇, 440.3).

Example 3 2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-4-methyl-pentanoicacid methyl ester

General procedure A was followed using 1 (23 mg, 0.05 mmol), D-leucinemethyl ester hydrochloride (47 mg, 0.25 mmol), and DIEA (45 μL, 0.25mmol) in EtOH (2 mL). Purification via flash chromatography (1%MeOH/CH₂Cl₂) afforded the product as a clear oil (19 mg, 0.04 mmol, 83%yield); R_(f)=0.22 (15% EtOAc/CH₂Cl₂); LRMS (m/z) [M+1]⁺454.3(calculated for C₂₄H₄₀NO₇, 454.3).

Example 4 {(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R, 3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-phenyl-aceticacid methyl ester

General procedure A was followed using 1 (37 mg, 0.08 mmol), D-phenylglycine methyl ester hydrochloride (83 mg, 0.40 mmol), and DIEA (72 μL,0.40 mmol) in EtOH (2 mL). Purification via flash chromatography (1%MeOH/CH₂Cl₂) afforded the product as a clear oil (32 mg, 0.07 mmol, 82%yield); R_(f)=0.41 (2% MeOH/CH₂Cl₂); LRMS (m/z) [M+1]⁺474.3 (calculatedfor C₂₆H₃₆NO₇, 474.3).

Example 5 (1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General procedure A was followed using 1 (55 mg, 0.12 mmol), D-valineamide hydrochloride (93 mg, 0.62 mmol), and DIEA (110 μL, 0.62 mmol) inEtOH (2 mL). Purification via flash chromatography (2% MeOH/CH₂Cl₂)afforded the product as a clear oil (42 mg, 0.10 mmol, 80% yield);R_(f)=0.19 (2% MeOH/CH₂Cl₂); LRMS (m/z) [M+1]⁺425.5 (calculated forC₂₂H₃₇N₂O₆, 425.5).

Example 6 (1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl ester

The compound in Example 4 (18 mg, 0.04 mmol) was dissolved intoanhydrous MeOH (1.5 mL) and treated with Pd—C (2 mg) under a H₂atmosphere. After 12 hours, the reaction was filtered through Celite andthe solvent removed in vacuo to afford the product as a clear oil (18mg, 0.04 mmol, 100% yield); R_(f)=0.21 (2% MeOH/CH₂Cl₂); LRMS (m/z)[M+1]⁺427.5 (calculated for C₂₂H₃₉N₂O₆, 427.5).

Example 7 (1-Hydroxymethyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General procedure A was followed using 1 (290 mg, 0.65 mmol), D-valinol(337 mg, 3.25 mmol), and DIEA (560 μL, 3.25 mmol) in EtOH (5 mL).Purification via flash chromatography (2% MeOH/CH₂Cl₂) afforded theproduct as a clear oil (200 mg, 0.49 mmol, 75% yield); R_(f)=0.26 (2%MeOH/CH₂Cl₂); LRMS (m/z) [M+1]⁺412.5 (calculated for C₂₂H₃₈NO₆, 412.5).

Example 8 2-{(3R, 4S, 5S, 6R )-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3,3-dimethyl-butyricacid methyl ester

General procedure A was followed using 1 (65 mg, 0.15 mmol), D-tBuglycine methyl ester hydrochloride (132 mg, 0.73 mmol), and DIEA (127μL, 0.73 mmol) in EtOH (8 mL). Purification via flash chromatography(10% EtOAc/CH₂Cl₂) afforded the product as a clear oil (10 mg, 0.02mmol, 15% yield); R_(f)=0.22 (10% EtOAc/CH₂Cl₂); LRMS (m/z) [M+1]⁺454.5(calculated for C₂₄H₄₀NO₇, 454.5).

Example 9 Cyclohexyl-2-{(3R, 4S, 5S, 6R )-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-aceticacid methyl ester

General procedure A was followed using 1 (65 mg, 0.15 mmol),D-cyclohexyl glycine methyl ester hydrochloride (207 mg, 0.73 mmol), andDIEA (127 μL, 0.73 mmol) in EtOH (7 mL). Purification via flashchromatography (10% EtOAc/CH₂Cl₂) afforded the product as a clear oil(20 mg, 0.04 mmol, 28% yield); R_(f)=0.22(10% EtOAc/CH₂Cl₂); LRMS (m/z)[M+1]⁺480.3 (calculated for C₂₆H₄₂NO₇, 480.3).

Example 10 2-{(3R, 4S, 5S, 6R )-5-Methoxy-4-[(2R,3R)-2-methyl-3-3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3-methyl-pentanoicacid methyl ester

General procedure A was followed using 1 (65 mg, 0.15 mmol),D-isoleucine methyl ester hydrochloride (132 mg, 0.73 mmol), and DEA(127 μL, 0.73 mmol) in EtOH (7 mL). Purification via flashchromatography (10% EtOAc/CH₂Cl₂) afforded the product as a clear oil(20 mg, 0.04 mmol, 30% yield); R_(f)=0.20 (10% EtOAc/CH₂Cl₂); LRMS(in/z) [M+1]⁺454.5 (calculated for C₂₄H₄₀NO₇, 454.5).

Example 11[1-(1-Carbamoyl-2-hydroxy-ethylcarbamoyl)-2-methyl-propyl]-carbamicacid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl]-oxiranyl-1-oxa-spiro[2.5]oct-6-ylester

General procedure A was followed using 1 (74 mg, 0.17 mmol),H-D-vS-NH₂.TFA (262 mg, 0.83 mmol), and DIEA (140 μL, 0.83 mmol) in EtOH(5 mL). Purification via HPLC (60% CH₃CN/H₂O) afforded the as a whitesolid (34 mg, 0.07 mmol, 40% yield); R_(f)=0.21 (5% MeOH/CH₂Cl₂); LRMS(m/z) [M+1]⁺512.5 (calculated for C₂₅H₄₂N₃O₈, 512.3).

Example 12 2-(3-{(3R, 4S, 5S, 6R )-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl}-ureido)-3-methyl-butyramide

(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylamine(3; PCT Publication No. WO 99/59987) was prepared according to thepublished procedure. To a solution of crude 3 (29 mg, 0.1 mmol), DIEA(21 mL, 0.1 mmol) and DMAP (2 mg) in CH₂Cl₂ (1.5 mL) cooled to 0° C. wasadded p-NO₂ phenyl chloroformate (25 mg, 0.12 mmol). After 45 minutes,the reaction was warmed to room temperature and a solution ofH-D-val-NH₂.HCl (40 mg, 0.15 mmol) in EtOH (1 mL) and DIEA (35 μL, 0.2mmol) was added.

The reaction was continued for 1 hour, then was concentrated in vacuo,taken up into EtOAc (15 mL), and washed with dilute HCl_(aq)(2×15 mL),H₂O (2×15 mL) and brine (15 mL). Purification via flash chromatography(5% MeOH/CH₂Cl₂) afforded the product as a white solid (6 mg, 0.014mmol, 13% yield from 3); R_(f)=0.12 (5% MeOH/CH₂Cl₂); LRMS (m/z)[M+1]⁺424.4 (calculated for C₂₂H₃₈N₃O₅, 424.4).

Example 13 N-Carbamoyl (ID#31) 3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General Procedure B, Part I was followed using 2 (41 mg, 0.11 mmol),PS-DCC (256 mg, 0.28 mmol) in DMF (1 mL) and H—RGD(Bn)S(OBn)P—NH₂.2TFA(37 mg, 0.04 mmol), NMM (4 μL, 0.04 mmol) in DMF (0.5 mL). Purificationvia HPLC (70% CH₃CN/H₂O/0.075% TFA) afforded the product as a whitefloculent solid (9.3 mg, 0.009 mmol, 17% yield); LRMS (m/z) [M+1]⁺1075.4(calculated for C₅₃H₇₅N₁₀O₁₄, 1075.5).

General Procedure, Part II was followed using the product in Part I (9.3mg, 0.009 mmol) and Pd/C (2 mg) in MeOH (1 mL), and a H₂ atmosphere (38psi) for 24 hours. Purification via HPLC (55% CH₃CN/H₂O/0.075% TFA)afforded the product as a white solid (5 mg, 0.006 mmol, 65% yield);LRMS (m/z) [M+1]⁺897.3 (calculated for C₃₉H₆₅N₁₀O₁₄, 897.5).

Example 14 N-Carbamoyl (ID#30) 3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General Procedure, Part I was followed using 2 (38 mg, 0.10 mmol) andPS-DCC (238 mg, 0.25 mmol) in DMF (1 mL), H—RGD(Bn)Y(OMe)RE(Bn)-NH₂.3TFA(35 mg, 0.03 mmol) and NMM (3 μL, 0.03 mmol) in DMF (0.5 mL).Purification via HPLC (70% CH₃CN/H₂O/0.075% TFA) afforded the product asa white floculent solid (4.0 mg, 0.002 mmol, 8% yield); LRMS (m/z)[M+2/2]⁺677.6 (calculated for C₆₆H₉₂N14O₁₇, 677.8).

General Procedure, Part II was followed using the product in Part I (3.0mg, 0.002 mmol) and Pd/C (2 mg) in MeOH (1 mL), under a H₂ atmosphere(38 psi) for 24 hours. Purification via HPLC (55% CH₃CN/H₂O/0.075% TFA)afforded the product as a white solid (3.3 mg, 0.0027 mmol, 94% yield);LRMS (m/z) [M+2/2]⁺588.5 (calculated for C₅₂H₈₂N14O₁₇, 588.7).

Example 15 N-Carbamoyl (ID#32) 3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General Procedure B, Part I was followed using 2 (38 mg, 0.10 mmol),PS-DCC (238 mg, 0.25 mmol), and HOBt (29 mg, 0.25 mmol) in DMF (1 mL),and H—RGD(Bn)NH₂.TFA (29 mg, 0.04 mmol) and NMM (4.8 μL, 0.04 mmol) inDMF (0.5 mL). Purification via HPLC (60% CH₃CN/H₂O/0.075% TFA) affordedthe product as a white solid (35 mg, 0.04 mmol, 44% yield); LRMS (m/z)801.2 (calculated for C₃₈H₅₇N₈O₁₁, 801.4).

General Procedure, Part II was followed using the product in Part 1 (35mg, 0.04 mmol) and Pd/C (2 mg) in MeOH (1 mL), under a H₂ atmosphere (38psi) for 24 hours. Purification via HPLC (50% CH₃CN/H₂O/0.075% TFA)afforded the product as a white solid (22 mg, 0.03 mmol, 71% yield);LRMS (m/z) 713.2 (calculated for C₃₁H₅₃N₈O₁₁, 713.4).

Example 16 N-Carbamoyl (ID#40) (3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General Procedure B, Part I was followed using 2 (65 mg, 0.17 mmol),PS—DCC (405 mg, 0.43 mmol), and HOBt (34 mg, 0.26 mmol) in DMF (1 mL),and H—RG(pyridyl)D-OMe (43 mg, 0.06 mmol) and NMM (7 μL, 0.06 mmol) inDMF (0.5 mL). Purification via HPLC (50% CH₃CN/H₂O/0.075% TFA) affordedthe product as a white solid (15 mg, 0.02 mmol, 34% yield); LRMS (m/z)773.2 (calculated for C₃₄H₅₅N₄O₁₀, 773.4).

The product of Part I (11 mg, 0.01 mmol) was dissolved into THF:MeOH:H₂O(2:1:1, 500 μL) and treated with LiOH.H₂O (1.2 mg, 0.02 mmol) for 2hours. The crude material was diluted with EtOAc (5 mL) and acidifiedwith dilute HCl (10 mL). The aqueous phase was washed with additionalEtOAc (2×5 mL), the combined organic extracts dried over Na₂SO₄ and thesolvent removed in vacuo. Purification via HPLC (30% CH₃CN/H₂O/0.075%TFA) afforded the product as a white solid (2 mg, 0.003 mmol, 19%yield). LRMS (m/z) 745.3 (calculated for C₃₃H₅₃N₄O₁₀, 745.4).

Example 17 N-Carbamoyl (ID#39) (3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General procedure B, Part I was followed using 2 (25 mg, 0.07 mmol) andPS-DCC (155 mg, 0.16 mmol) in DMF (1 mL), and H—PLGMWAG-NH₂ (20 mg, 0.03mmol) and NMM (3 μL, 0.03 mmol) in DMF (0.5 mL). Purification via HPLC(70% CH₃CN/H₂O/0.075% TFA) afforded the product as a white solid (1.4mg, 0.001 mmol, 5% yield); LRMS (m/z) [M+1]+1095.6 (calculated forC₅₃H₇₉N₁₀O₁₃S, 1095.6).

Example 18 N-Carbamoyl (ID#26) (3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General Procedure B, Part I was followed using 2 (69 mg, 0.18 mmol),PS-DCC (429 mg, 0.45 mmol) and HOBt (21 mg, 0.18 mmol) in DMF (1 mL),and H—PL(N—Me)G-OMe (31 mg, 0.07 mmol) and NMM (8 μL, 0.07 mmol) in DMF(0.5 mL). Purification via HPLC (70% CH₃CN/H₂O/0.075% TFA) afforded theproduct as a white solid (27 mg, 0.04 mmol, 59% yield); LRMS (m/z)[M+1]+679.4 (calculated for C₃₄H₅₅N₄O₁₀, 679.4).

The product of Part I (27 mg, 0.04 mmol) was dissolved into THF:MeOH:H₂O(2:1:1, 1.5 mL) and treated with LiOH—H₂O (4 mg, 0.10 mmol) for 1 hour.The solution was acidified to pH 3 using 0.1 N HCL, and the MeOH and THFremoved in vacuo. Purification via HPLC (60% CH₃CN/H₂O/0.075% TFA)afforded the product as a white solid (6 mg, 0.01 mmol, 23% yield). LRMS(m/z) 665.4 (calculated for C₃₃H₅₃N₄O₁₀, 665.4).

Example 19 N-Carbamoyl (ID#27) (3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester

General Procedure B, Part I was followed using 2 (44 mg, 0.12 mmol),PS-DCC (276 mg, 0.29 mmol) and HOBt (27 mg, 0.23 mmol) in DMF (1 mL),and H-PLG-OMe (20 mg, 0.05 mmol) and NMM (5 μL, 0.05 mmol) in DMF (0.5mL). Purification via HPLC (90% CH₃CN/H₂O/0.075% TFA) afforded theproduct as a white solid (13 mg, 0.02 mmol, 43% yield); LRMS (m/z)[M+1]⁺664.4 (calculated for C₃₄H₅₂N₄O₁₀, 664.4).

The product in Part I (27 mg, 0.04 mmol) was dissolved into THF:MeOH:H₂O(2:1:1, 790 μL) and treated with LiOH.H₂O (1.2 mg, 0.03 mmol) for 2hours. The solution was acidified to pH 3 using 0.1 N HCl, and the MeOHand THF removed in vacuo. Purification via HPLC (90% CH₃CN/H₂O/0.075%TFA) afforded the product as a white solid (1.8 mg, 0.003 mmol, 15%yield). LRMS (m/z) 650.4 (calculated for C₃₂H₅₀N₄O₁₀, 650.4).

Example 20 (ID#24)-(2R-{(3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester

General Procedure C was followed using the compound in Example 7 (189mg, 0.46 mmol), Ac-PLGMWA-OH (329 mg, 0.46 mmol), DMAP (84 mg, 0.69mmol) and DIC (72 μL, 0.46 mmol) in CH₂Cl₂ (5 mL). After 18 hours, thesolvent was removed in vacuo and purification via flash chromatography(2% MeOH/CH₂Cl₂) afforded the product as a white solid (357 mg, 0.32mmol, 70% yield); R_(f)=0.18 (5% MeOH/CH₂Cl₂); LRMS (m/z) [M+1]⁺1110.3(calculated for C₅₆H₈₅N₈O₁₃S, 1110.3).

Example 21 (ID#36)-(2R-{(3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester

General Procedure C was followed using the compound in Example 7 (61 mg,0.15 mmol), Ac-PLGMG-OH (92 mg, 0.18 mmol), DMAP (22 mg, 0.18 mmol) andDIC (28 μL, 0.18 mmol) in CH₂Cl₂ (2 mL). After 7 hours, the solvent wasremoved in vacuo and purification via flash chromatography (3%MeOH/CH₂Cl₂) afforded the product as a white solid (61 mg, 0. mmol, 45%yield); R_(f)=0.20 (5% MeOH/CH₂Cl₂); LRMS (m/z) [M+1]+909.7 (calculatedfor C₄₄H₇₃N₆O₁₂S, 909.5).

Example 22 (ID#37)-(2R-{(3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester

General Procedure C was followed using the compound in Example 7 (79 mg,0.19 mmol), Fmoc-MWA-OH (121 mg, 0.19 mmol) and DMAP (4 mg, 0.03 mmol)and DIC (30 μL, 0.19 mmol) in CH₂Cl₂ (2 mL). After 11 hours, the solventwas removed in vacuo and purification via flash chromatography (2%MeOH/CH₂Cl₂) afforded the product as a white solid (128 mg, 0.12 mmol,65% yield); LRMS (m/z) [M+1]⁺1022.9 (calculated for C₄₄H₇₃N₆O₁₂S,1022.5).

The product from General Procedure C (above) (54 mg, 0.05 mmol) wasdissolved into anhydrous CH₂Cl₂ (3 mL) cooled to 0° C., then treatedwith a gentle stream of NH_(3(g)) for 15 minutes. The reaction wassealed and continued at 0° C. for 36 hours. The solvent was removed invacuo, and the crude residue acidified with CH₃CN/H₂O(0.075% TFA) (5mL). Purification via HPLC (70% CH₃CN/H₂O/0,075% TFA) afforded theproduct as a white solid (2 mg, 0.003 mmol, 5% yield); LRMS (m/z)[M+1]⁺800.6 (calculated for C₄₁H₆₂N₅O₉S, 800.5).

Example 23 (ID#38)-(2R-{(3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester

General Procedure C was followed using the compound in Example 7 (76 mg,0.18 mmol), Fmoc-MG-OH (79 mg, 0.18 mmol) and DMAP (4 mg, 0.03 mmol) andVDIC (29 μL, 0.18 mmol) in CH₂Cl₂ (2 mL). After 10 hours, the solventwas removed in vacuo and purification via flash chromatography (2%MeOH/CH₂Cl₂) afforded the product as a white solid (128 mg, 0.12 mmol,65% yield); LRMS (m/z) [M+1]⁺822.6 (calculated for C₄₄H₆₀N₃O₁₀S, 822.5).

The product from General Procedure C (above) (42 mg, 0.05 mmol) wasdissolved into anhydrous CH₂Cl₂ (3 mL) cooled to 0° C., then treatedwith a gentle stream of NH_(3(g)) for 15 minutes. The reaction wassealed and continued at 0° C. for 36 hours. The solvent was removed invacuo, and the crude residue acidified with CH₃CN/H₂O (0.075% TFA) (5mL). Purification via HPLC (70% CH₃CN/H₂O/0.075% TFA) afforded theproduct as a white solid (2 mg, 0.003 mmol, 5% yield); LRMS (m/z)[M+1]⁺600.4 (calculated for C₂₉H₅₀N₃O₈S, 600.4).

Example 24 2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3-methyl-butyricacid

The compound in Example 2 (9 mg, 0.02 mmol) was dissolved intoTHF:MeOH:H₂O (1 mL) and treated with LiOH.H₂O (2 mg, 0.05 mmol). After 2hours, the reaction was partitioned between EtOAc (5 mL) and dilute HCl(5 mL). The organic phase was dried over Na₂SO₄ and the solvent removedin vacuo. Purification via HPLC (85% CH₃CN/H₂O/0.075% TFA) afforded theproduct as a white solid (0.58 mg, 0.001 mmol, 6% yield); LRMS (m/z)[M+1]⁺426.4 (calculated for C₂₂H₃₆NO₇, 426.5).

Example 25 (ID#34)-(2R-{(3R, 4S, 5S, 6R)5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester

General Procedure C was followed using the compound in Example 7 (41 mg,0.10 mmol), Ac-PLGMG-OH (63 mg, 0.12 mmol), DMAP (15 mg, 0.12 mmol) andDIC (19 μL, 0.12 mmol) in CH₂Cl₂ (2 mL). After 7 hours, the solvent wasremoved in vacuo and purification via flash chromatography (3%MeOH/CH₂Cl₂) afforded the product as a white solid (43 mg, 0.05 mmol,47% yield); R_(f)=0.21 (5% MeOH/CH₂Cl₂); LRMS (m/z) [M+1]⁺923.7(calculated for C₄₅H₇₅N₆O₁₂S, 923.5).

Example 26

The angiogenesis inhibitor compounds of the invention were tested fortheir ability to modulate human endothelial cell growth and for theirability to modulate the activity of MetAP2. The MetAP2 enzyme assay wasperformed essentially as described in Turk, B. et al. (1999) Chem. &Bio. 6: 823-833, the entire contents of which are incorporated herein byreference. The bovine aortic endothelial cell growth assay (Baec assay)was performed essentially as described in Turk, B. et al. (supra), theentire contents of which are incorporated herein by reference.

For the human endothelial cell growth assay, human umbilical veinendothelial cells (HUVEC) were maintained in Clonetics endothelialgrowth medium (EGM) in a 37° C. humidified incubator. Cells weredetached with trypsin and pelleted by centrifugation at 300×g for 5minutes at room temperature. HUVEC were added to 96-well plates at 5,000cells/well. After incubating for 6 hours, the medium was replaced with0.2 ml fresh EGM supplemented with 0.5 μM bFGF and the desiredconcentration of test angiogenesis inhibitor compound. Test angiogenesisinhibitor compounds were initially dissolved in ethanol at stockconcentrations of either 10 mM or 0.1 mM, and subsequently diluted inEGM to obtain concentrations from 1 μM to 10 μM. After 48 hours at 37°C., the medium was replaced with fresh bFGF-supplemented EGM and testangiogenesis inhibitor compound. Following incubation for an additional48 hours at 37° C. MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) wasadded to 1 mg/ml. After 2-4 hours at 37° C. the medium was replaced with0.1 ml/well isopropanol. The plates were placed on a shaker for 15minutes at room temperature and analyzed in a Labsystems Multiskan platespectrophotometer at an optical density of 570 nm.

The results of the assays, set forth below in Tables I-III, demonstratethat the angiogeness inhibitor compounds of the invention have excellentMetAP2 inhibitory activity and are able to inhibit endothelial cellgrowth at the picomolar range.

TABLE I MetAP2 Assay Example IC₅₀ (nM) 1 4.7 2 2 3 5.5 4 2.7 13 2.9 144000 17 16.7

TABLE II Huvec Assay Example IC₅₀ (pM) 1 18 2 40 3 38 4 36 5 93 13 (>10μM) 14 (>10 μM) 15 (>10 μM) 17 (95 nM) 18 (>100 nM) 19 (>100 nM) 24 5444

TABLE III Baec Assay Example IC₅₀ (pM) 1 17 2 48 3 118 4 35 5 46 6 220 7128 8 313 9 165 10 179 11 (>100 nM) 16 (>100 nM) 19 (>100 nM) 22 326 23207The identity of the angiogenesis inhibitor compounds used in each of theexperiments is shown in Tables IV and V below.

TABLE IV

Example ID # Sequence 13 31 X-GlyArgGlyAspSerPro-NH₂ 14 30X-GlyArgGlyAspTyr(OMe)Arg Glu-NH₂ 15 32 X-GlyArgGlyAsp-NH₂ 16 40X-Gly-Arg-Gly-(3-amino-3-pyridylpropionic acid) 17 39X-GlyProLeuGlyMetTrpAlaGly-NH₂ 18 26 X-GlyProLeuSar-OH 19 27X-GlyProLeuGly-OH

TABLE V

Example ID # Sequence 20 24 Ac-ProLeuGly-MetTrpAla-Y 21 36Ac-ProLeuGlyMetGly-Y 22 37 H-MetTrpAla-Y 23 38 H-MetGly-Y 25 34Ac-ProLeuGlyMetAla-Y

Example 27

The angiogenesis inhibitor compounds of the invention were also testedfor their ability to modulate angiogenesis using the rat aortic ringassay (RARA). For this assay, male Sprague-Dawley rats weighing 125-150grams were sacrificed by an intramuscular followed by a cardiacinjection of catemine, submerged in ethanol, and transferred to asterile culture hood for dissection. Following a mid-abdominal incisionextending to the thorax, the ribs were resected to expose the thoraciccavity. The heart and lungs were moved aside to locate the thoracicaorta. The segment of the aorta distal to the aortic arch and extendingto the diaphragm was removed and placed immediately in MCDB-131 mediumcontaining 4 mM L-glutamine and 50 μg/ml gentamycin (buffer A). Buffer Awas gently pipeted through the aorta with a pipetman to remove blood.The fibrous tissue was surrounding the aorta was carefully removed andthe aorta was sliced into 1-2 mm sections with sterile razor blades. Thesections were collected in 50 ml conical tubes and washed five timeswith fresh buffer A.

Twenty four-well culture plates were coated with 0.2 ml autoclaved 1.5%agarose in buffer A. After the agarose solidified, the wells were filledwith 1 ml buffer A. One aortic segment was submerged in each well andincubated in a 35.5° C./5% CO₂ humidified incubator for 10 days. BufferA was replaced every 2 days. The aortic segments were collected in 50 mlconical tubes and washed five times with fresh buffer A.

One milliliter of autoclaved 1.5% agarose in buffer A was added to eachwell of a twenty four-well culture plate and allowed to solidify.Agarose wells were cut from the solidified agarose withethanol-sterilized cork borers and placed in twelve-well culture plates.Collagen solution was prepared by mixing 12.8 ml CollaborativeBiomedical Products rat tail type I collagen with 2 ml 7.5× Dulbecco'sModified Eagle's Medium adjusted to pH 7 with approximately 0.2 ml 1NNaOH. One hundred microliters of collagen solution was added to the baseof the agarose wells and allowed to set in a 35.5° C./5% CO₂ humidifiedincubator. Three hundred microliters of collagen solution supplementedwith 0.5 nM bFGF and the desired concentration of test compound wasadded and an aortic segment was submerged within the upper collagenlayer. Test compounds were initially dissolved in ethanol at a stockconcentration of 0.1 mM, and subsequently diluted in buffer A andfinally collagen solution to obtain concentrations from 0.1 nM to 100nM. After the collagen set, the agarose wells were surrounded by 1.5 mlbuffer A supplemented with 0.5 nM bFGF and the desired concentration oftest angiogenesis inhibitor compound. The buffer surrounding the wellswas replaced every 48 hours. After seven days, the vessels protrudingfrom the aortic rings were counted under a microscope.

The results of this assay, set forth herein in FIG. 1, demonstrate thatthe angiogeness inhibitor compounds of the invention have excellentangiogenesis inhibitory activity (much better compared to theangiogenesis inhibitory activity of TNP-470).

Example 28

The plasma stability of the angiogenesis inhibitor compounds of theinvention was also tested in human plasma at 37° C. (at a 20 μMconcentration of the angiogenesis inhibitor compound). The matrix minusthe test angiogenesis inhibitor compound was equilibrated at 37° C. for3 minutes. The assay was initiated by addition of the test compound, andthe reaction mixture was incubated in a shaking water bath at 37° C.Aliquots (100 μL) were withdrawn in triplicate at 0, 60, and 120 minutesand combined with 200 μL of ice cold acetonitrile to terminate thereaction. The supernatant was analysed by Liquid Chromatography/Massspectrometry (LC/MS) using a four point standard curve to obtain percentrecovery.

The parent compound remaining (relative to time 0) in the incubationmixture was plotted as a function of time; a first order exponentialequation was fit to the observed data, and the elimination half-livesassociated with the disappearance of the test angiogenesis inhibitorcompound were determined. Results are shown below in Table VI.

TABLE VI Percent Recovery (Remaining) Test 120 T_(1/2) Compound 0 min 15min 30 min 60 min 90 min min (min)  5 100 108 103  97 129 107 ≧500 20110 114 114 120 106 103 ≧500

The stability of the angiogenesis inhibitor compounds in livermicrosomes was also tested. The test angiogenesis inhibitor compound ata concentration of 3 μM was incubated with male rat liver microsomes(0.5 mg/mL protein) in the presence of a NADPH regenerating system (1 mMNADP, 10 mM G6P, 1 U/mL G6PdH2, 3 mM MgCl₂) in 100 mM Phosphate bufferat pH 7.4 at 37° C. Incubations were performed in the presence andabsence of 1 μM epoxide hydrolase (EH) inhibitorN-cyclohexyl-N′-(3-phenylpropyl)urea (M. Morisseau et al. (1999) PNAS96:8849-54) and quantitative analysis performed using LC/MS/MS.

The parent compound remaining (relative to time 0) in the incubationmixture was plotted as a function of time; a first order exponentialequation was fit to the observed data, and the elimination half-livesassociated with the disappearance of the test compound were determined.Results are shown below in Table VII.

TABLE VII Test T_(1/2) T_(1/2) Compound (without EH inhibitor) mins(with EH inhibitor) mins TNP-470 >1.0 >1.0 5 11.9 ± 1 12.1 ± 1

Example 29

The efficacy of the compound described above in example 5 was evaluatedusing a collagen-induced arthritis model. Briefly, syngeneic 8-week-oldfemale Louvain rats were immunized intradermally under ether anesthesiaon Day 0 with 0.5 mg chick collagen II (Genzyme, Boston, Mass.)solubilized in 0.1M acetic acid and emulsified in Incomplete Freund'sAdjuvant (Difco, Detroit, Mich.). Using this method, more than 90% ofrats typically develop synovitis in both hind limbs 10-14 days postimmunization. Rats demonstrating definite arthritis (47 total) wereentered into the study and randomly assigned to one of four protocols.The compound of example 5 was dissolved in a combination of ethanol anddistilled water. The rats were dosed commencing on day 10 postimmunization as follows: (1) control group: vehicle only daily viaintravenous injection (iv) (12 rats), (2) high dose group: 30 mg/kg ofthe compound of example 5 iv every other day (12 rats), (3) low dosegroup: 15 mg/kg of the compound of example 5 iv every day (12 rats) and(4) oral group: 100 mg/kg of the compound of example 5 every other dayby gavage (11 rats).

Daily clinical evaluations of each paw were graded on an integer scaleranging from 0 to 4, where a score of 0 indicates a normal, unaffectedlimb, and 4 indicates severe joint destruction. The clinical score isthe sum of the four limb scores, and has a maximum possible value of 16.Collagen induced arthritis typically develops only in the hind limbs;therefore, a score of 6 to 8 represents severe arthritis.

Radiologic evaluations of the hind limbs were performed by aninvestigator blinded to the treatment protocol. The score assigned toeach limb was based on (1) the degree of soft tissue swelling, (2)narrowing of the space between joints, (3) degree of periosteal new boneformation and (4) the presence of erosions or ankylosis. Each limb wasscored on a scale of 1 to 3, with 1 representing a normal limb and 3representing severe joint destruction. Each rat, thus, had a maximumpossible radiographic score of 6 (sum of both hind limbs).

The results of this study are set forth in FIG. 2, which presents themean daily clinical score for each of the four groups of rats over the19 day period following induction of clinical arthritis (day 10). Theresults indicate that each treatment group received a significanttherapeutic benefit from the compound of example 5. Consistent withthese observations, the mean radiological evaluation scores were 2.2 forthe control group, 0.6 for the high dose group, 0.6 for the low dosegroup and 1.6 for the oral group. Thus, each group treated with thecompound of example 5 received a significant benefit with regard tojoint destruction compared to the control group.

Example 30

The compound of example 5 was also evaluated against a panel of cancercell lines (Alley, M. C. et al. (1998) Cancer Research 48: 589-601;Grever, M. R., et al. (1992) Seminars in Oncology, Vol. 19, No. 6, pp622-638; Boyd, M. R., and Paull, K. D. (1995) Drug Development Research34: 91-109). The human tumor cell lines of the cancer screening panelwere grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mML-glutamine. Cells were inoculated into 96 well microtiter plates in 100μL at plating densities ranging from 5,000 to 40,000 cells/welldepending on the doubling time of individual cell lines. After cellinoculation, the microtiter plates were incubated at 37° C., 5% CO₂, 95%air and 100% relative humidity for 24 hours prior to addition ofexperimental drugs.

After the 24 hour incubation period, two plates of each cell line werefixed in situ with TCA, to represent a measurement of the cellpopulation for each cell line at the time of drug addition (Tz).Experimental drugs were solubilized in dimethyl sulfoxide at 400-foldthe desired final maximum test concentration and stored frozen prior touse. At the time of drug addition, an aliquot of frozen concentrate wasthawed and diluted to twice the desired final maximum test concentrationwith complete medium containing 50 μg/ml gentamicin. Additional four,10-fold or ½ log serial dilutions were made to provide a total of fivedrug concentrations plus control. Aliquots of 100 μl of these differentdrug dilutions were added to the appropriate microtiter wells alreadycontaining 100 μl of medium, resulting in the required final drugconcentrations.

Following drug addition, the plates were incubated for an additional 48hours at 37° C., 5% CO₂, 95% air, and 100% relative humidity. Foradherent cells, the assay was terminated by the addition of cold TCA.Cells were fixed in situ by the gentle addition of 50 μl of cold 50%(w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at4° C. The supernatant was discarded, and the plates were washed fivetimes with tap water and air dried. Sulforhodamine B (SRB) solution (100μl) at 0.4% (w/v) in 1% acetic acid were added to each well, and plateswere incubated for 10 minutes at room temperature. After staining,unbound dye was removed by washing five times with 1% acetic acid andthe plates were air dried. Bound stain was subsequently solubilized with10 mM trizma base, and the absorbance was read on an automated platereader at a wavelength of 515 nm. For suspension of the cells, themethodology used was the same except that the assay was terminated byfixing settled cells at the bottom of the wells by gently adding 50 μlof 80% TCA (final concentration, 16% TCA). Using the seven absorbancemeasurements [time zero, (Tz), control growth, (C), and test growth inthe presence of drug at the five concentration levels (Ti)], thepercentage growth was calculated at each of the drug concentrationslevels.

Percentage growth inhibition was calculated as:[(Ti−Tz)/(C−Tz)]×100 for concentrations for which Ti>/=Tz[(Ti−Tz)/Tz]×100 for concentrations for which Ti<Tz.

Growth inhibition of 50% (GI₅₀) was calculated from[(Ti−Tz)/(C−Tz)]×100=50, which is the drug concentration resulting in a50% reduction in the net protein increase (as measured by SRB staining)in control cells during the drug incubation. The GI₅₀ was calculated foreach of the cell lines if the level of activity is reached; however, ifthe effect was not reached or is exceeded, the value for that parameteris expressed as greater or less than the maximum (10⁻⁴ M) or minimum(10⁻⁸ M) concentration tested.

TABLE VIII Effect of the compound of example 5 on tumor cell line panelCell line Tumor type GI₅₀ (moles liter⁻¹) HL-60(TB) Leukemia 2.17 × 10⁻⁵K-562 Leukemia 6.44 × 10⁻⁵ MOLT-4 Leukemia 3.56 × 10⁻⁵ RPMI-8226Leukemia   <1 × 10⁻⁸ SR Leukemia   <1 × 10⁻⁸ EKVX Non-Small Cell Lung2.08 × 10⁻⁵ HOP-62 Non-Small Cell Lung   <1 × 10⁻⁸ HOP-92 Non-Small CellLung 3.39 × 10⁻⁵ NCI-H226 Non-Small Cell Lung 7.91 × 10⁻⁷ NCI-H23Non-Small Cell Lung 6.34 × 10⁻⁶ NCI-H322M Non-Small Cell Lung 4.68 ×10⁻⁸ NCI-H460 Non-Small Cell Lung   <1 × 10⁻⁸ NCI-H522 Non-Small CellLung 1.29 × 10⁻⁵ COLO 205 Colon   <1 × 10⁻⁸ HCT-116 Colon   <1 × 10⁻⁸HCT-15 Colon 7.13 × 10⁻⁶ HT29 Colon 1.61 × 10⁻⁵ KM12 Colon   <1 × 10⁻⁸SW-620 Colon   >1 × 10⁻⁴ SF-268 CNS 2.61 × 10⁻⁵ SF-295 CNS   <1 × 10⁻⁸SF-539 CNS 2.06 × 10⁻⁵ SNB-19 CNS   <1 × 10⁻⁸ SNB-75 CNS 9.09 × 10⁻⁵MALME-3M Melanoma 5.31 × 10⁻⁸ M14 Melanoma   <1 × 10⁻⁸ SK-MEL-2 Melanoma  >1 × 10⁻⁴ SK-MEL-28 Melanoma 5.96 × 10⁻⁶ SK-MEL-5 Melanoma   >1 × 10⁻⁴UACC-257 Melanoma 1.48 × 10⁻⁶ UACC-62 Melanoma   <1 × 10⁻⁸ IGR-OV1Ovarian   <1 × 10⁻⁸ OVCAR-3 Ovarian 4.18 × 10⁻⁵ OVCAR-4 Ovarian 3.66 ×10⁻⁵ OVCAR-5 Ovarian 1.35 × 10⁻⁸ OVCAR-8 Ovarian 1.84 × 10⁻⁵ SK-OV-3Ovarian 7.37 × 10⁻⁶ 786-0 Renal 1.61 × 10⁻⁵ A498 Renal   >1 × 10⁻⁴ ACHNRenal   <1 × 10⁻⁸ CAKI-1 Renal   <1 × 10⁻⁸ RXF 393 Renal 4.02 × 10⁻⁵SN12C Renal   <1 × 10⁻⁸ TK-10 Renal 5.43 × 10⁻⁸ PC-3 Prostate 1.80 ×10⁻⁵ DU-145 Prostate   <1 × 10⁻⁸ MCF7 Breast 1.24 × 10⁻⁵ NCI/ADR-RESBreast 3.42 × 10⁻⁵ MDA-MB-231/ATCC Breast   <1 × 10⁻⁸ HS 578T Breast1.15 × 10⁻⁶ MDA-N Breast 1.58 × 10⁻⁶Results

The results of the cell line screen, presented in Table VIII, show thatthe compound of example 5 has a significant inhibitory effect on a widevariety of tumor cell lines. The results also show that certain celllines are much more sensitive to the compound of example 5 than areothers, indicating that this compound is selective for certain celllines.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of treating a subject having melanoma, comprisingadministering to said subject an effective amount of a compoundcomprising the structure of Formula I,

wherein A is a Met-AP2 inhibitory core; W is O or NR₂; R₁ and R₂ areeach, independently, hydrogen or alkyl; X is alkylene or substitutedalkylene; n is 0 or 1; R₃ and R₄ are each, independently, hydrogen,substituted or unsubstituted alkyl, substituted or unsubstituted aryl orsubstituted or unsubstituted heteroaryl; or R₃ and R₄, together with thecarbon atom to which they are attached, form a carbocyclic orheterocyclic group; or R₃ and R₄ together form an alkylene group; Z is—C(O)— or alkylene-C(O)—; and P is a peptide comprising from 1 to about100 amino acid residues attached at its amino terminus to Z or a groupOR₅ or N(R₆)R₇, wherein R₅, R₆ and R₇ are each, independently, hydrogen,alkyl, substituted alkyl, azacycloalkyl or substituted azacycloalkyl; orR₆ and R₇, together with the nitrogen atom to which they are attached,form a substituted or unsubstituted heterocyclic ring structure; or Z is—O—, —NR₈—, alkylene-O— or alkylene-NR₈—, where R₈ is hydrogen; and P ishydrogen or a peptide consisting of from 1 to about 100 amino acidresidues attached at its carboxy terminus to Z; and pharmaceuticallyacceptable salts thereof.
 2. The method of claim 1, wherein at least oneof R₁, R₃ and R₄ is a substituted or unsubstituted alkyl group.
 3. Themethod of claim 2, wherein at least one of R₁, R₃ and R₄ is asubstituted or unsubstituted normal, branched or cyclic C₁-C₆ alkylgroup.
 4. The method of claim 3, wherein at least one of R₁, R₃ and R₄is a normal or branched C₁-C₄ alkyl group.
 5. The method of claim 1,wherein one of R₃ and R₄ is a substituted or unsubstituted aryl group, asubstituted or unsubstituted heteroaryl group, a substituted orunsubstituted heteroarylalkyl group, or a substituted or unsubstitutedaryl alkyl group.
 6. The method of claim 5, wherein one of R₃ and R₄ isselected from the group consisting of phenyl, naphthyl, indolyl,imidazolyl, pyridyl, benzyl, naphthylmethyl, indolylmethyl,imidazolylmethyl and pyridylmethyl.
 7. The method of claim 1, wherein nis 1 and X is C₁-C₆-alkylene.
 8. The method of claim 7, wherein X ismethylene or ethylene.
 9. The method of claim 1, wherein Z isC₁-C₆-alkylene-C(O)—.
 10. The method of claim 9, wherein Z ismethylene-C(O)— or ethylene-C(O)—.
 11. The method of claim 1, wherein atleast one of R₆ and R₇ is alkyl, substituted alkyl, substituted orunsubstituted azacycloalkyl or substituted or unsubstitutedazacycloalkylalkyl.
 12. The method of claim 11, wherein at least one ofR₆ and R₇ is an azacycloalkyl group having an N-alkyl substituent. 13.The method of claim 12, wherein the N-alkyl substituent is a C₁-C₄-alkylgroup.
 14. The method of claim 13, wherein the N-alkyl substituent is amethyl group.
 15. The method of claim 1, wherein R₆ and R₇, togetherwith the nitrogen atom to which they are attached, form a substituted orunsubstituted five or six-membered aza- or diazacycloalkyl group. 16.The method of claim 15, wherein R₆ and R₇, together with the nitrogenatom to which they are attached, form a substituted or unsubstitutedfive or six-membered diazacycloalkyl group which includes an N-alkylsubstituent.
 17. The method of claim 16, wherein the N-alkyl substituentis a C₁-C₄-alkyl group.
 18. The method of claim 17, wherein the N-alkylsubstituent is a methyl group.
 19. The method of claim 1, wherein P isNH₂ or one of the groups shown below:


20. A method of treating a subject having melanoma, comprisingadministering to said subject an effective amount of a compoundcomprising the structure of Formula XV,

wherein A is a MetAP-2 inhibitory core; W is O or NR; each R is,independently, hydrogen or alkyl; Z is —C(O)— or -alkylene-C(O)—; P isNHR, OR or a peptide consisting of one to about one hundred amino acidresidues connected at the N-terminus to Z; Q is hydrogen, linear,branched or cyclic alkyl or aryl, provided that when P is —OR, Q is nothydrogen; or Z is -alkylene-O— or alkylene-N(R)—; P is hydrogen or apeptide consisting of from one to about one hundred amino acid residuesconnected to Z at the carboxyl terminus; Q is hydrogen, linear, branchedor cyclic alkyl or aryl, provided that when P is hydrogen, Q is nothydrogen; and pharmaceutically acceptable salts thereof.
 21. The methodof claim 20, wherein Z is —C(O)— or C₁-C₄-alkylene-C(O)—.
 22. The methodof claim 21, wherein Z is —C(O)— or C₁-C₂-alkylene-C(O)—.
 23. The methodof claim 21, wherein Q is linear, branched or cyclic C₁-C₆-alkyl, phenylor naphthyl.
 24. The method of claim 23, wherein Q is isopropyl, phenylor cyclohexyl.
 25. The method of claim 20, wherein Z isC₁-C₆-alkylene-O— or C₁-C₆-alkylene-NR—.
 26. The method of claim 25,wherein Z is C₁-C₄-alkylene-O— or C₁-C₄-alkylene-NH—.
 27. The method ofclaim 26, wherein Z is C₁-C₂-alkylene-O— or C₁-C₂-alkylene-NH.
 28. Themethod of claim 25, wherein Q is linear, branched or cyclic C₁-C₆-alkyl,phenyl or naphthyl.
 29. The method of claim 28, wherein Q is isopropyl,phenyl or cyclohexyl.
 30. The method of claim 20, wherein each R is,independently, hydrogen or linear, branched or cyclic C₁-C₆-alkyl. 31.The method of claim 30, wherein each R is, independently, hydrogen orlinear or branched C₁-C₄-alkyl.
 32. The method of claim 31, wherein eachR is, independently, hydrogen or methyl.
 33. The method of claim 32,wherein each R is hydrogen.
 34. The method of claim 20, wherein A is ofFormula II,

wherein R_(1′) is hydrogen or alkoxy; R_(2′) is hydrogen or hydroxy;R_(3′) is hydrogen or alkyl; and D is linear or branched alkyl orarylalkyl; or D is of the structure


35. The method of claim 34, wherein R_(1′) is C₁-C₄-alkoxy.
 36. Themethod of claim 35, wherein R_(1′) is methoxy.
 37. The method of claim34, wherein R_(3′) is hydrogen or C₁-C₄-alkyl.
 38. The method of claim37, wherein R_(3′) is methyl.
 39. The method of claim 34, wherein D islinear, branched or cyclic C₁-C₆-alkyl; or aryl-C₁-C₄-alkyl.
 40. Themethod of claim 20, wherein A is selected from the group consisting of

wherein p is an integer from 0 to 10; R_(1″) is hydrogen, —OH orC₁-C₄-alkoxy; X″ is a leaving group; and R₂″ is H, OH, amino,C₁-C₄-alkylamino or di(C₁-C₄-alkyl)amino.
 41. The method of claim 34,wherein A is of the formula


42. The method of claim 20, wherein P comprises from 1 to about 20 aminoacid residues.
 43. The method of claim 34, wherein P comprises an aminoacid sequence which is a substrate for a matrix metalloprotease.
 44. Themethod of claim 43, wherein the matrix metalloprotease is selected fromthe group consisting of MMP-2, MMP-1, MMP-3, MMP-7, MMP-8, MMP-9,MMP-12, MMP-13 and MMP-26.
 45. The method of claim 44, wherein thematrix metalloprotease is MMP-2 or MMP-9.
 46. The method of claim 45,wherein P comprises the sequence -Pro-Leu-Gly-Xaa-, wherein Xaa is anaturally occurring amino acid residue.
 47. The method of claim 46,wherein P comprises a sequence selected from the group consisting ofPro-Cha-Gly-Cys(Me)-His (SEQ ID NO:2); Pro-Gin-Gly-Ile-Ala-Gly-Gln-D-Arg(SEQ ID NO:3); Pro-Gln-Gly-Ile-Ala-Gly-Trp (SEQ ID NO:4);Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg (SEQ ID NO:5);Pro-Leu-Gly-Met-Trp-Ser-Arg (SEQ ID NO:35);Pro-Leu-Gly-Leu-Trp-Ala-D-Arg (SEQ ID NO:6); Pro-Leu-Ala-Leu-Trp-Ala-Arg(SEQ ID NO:7); Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:8);Pro-Leu-Ala-Tyr-Trp-Ala-Arg (SEQ ID NO:9); Pro-Tyr-Ala-Tyr-Trp-Met-Arg(SEQ ID NO:10); Pro-Cha-Gly-Nva-His-Ala (SEQ ID NO:11); Pro-Leu-Ala-Nva(SEQ ID NO:12); Pro-Leu-Gly-Leu (SEQ ID NO:13); Pro-Leu-Gly-Ala (SEQ IDNO:14); Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (SEQ ID NO:15);Pro-Cha-Ala-Abu-Cys(Me)-His-Ala (SEQ ID NO:16);Pro-Cha-Ala-Gly-Cys(Me)-His-Ala (SEQ ID NO:17);Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu (SEQ ID NO:18);Pro-Lys-Pro-Leu-Ala-Leu (SEQ ID NO:19);Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met (SEQ ID NO:20);Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:21);Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:22); andArg-Pro-Lys-Pro-Leu-Ala-Nva-Trp (SEQ ID NO:23).
 48. A method of treatinga subject having melanoma, comprising administering to said subject aneffective amount of a compound comprising the structure of the formula,

wherein W is O or NR′″; each R′″ is, independently hydrogen or aC₁-C₄-alkyl; Q is hydrogen; linear, branched or cyclic C₁-C₆-alkyl; oraryl; R_(1′″) is hydroxy, C₁-C₄-alkoxy or halogen; Z is —C(O)— orC₁-C₄-alkylene; P is NHR′″, OR′″, or a peptide comprising 1 to 100 aminoacid residues attached to Z at the N-terminus; or Z is alkylene-O oralkylene-NR′″; and P is hydrogen or peptide comprising 1 to 100 aminoacid residues attached to Z at the C-terminus; or a pharmaceuticallyacceptable salt thereof; provided that when P is hydrogen, NHR′″ orOR′″, Q is not hydrogen.
 49. The method of claim 48, wherein W is O orNH; Z is alkylene-O or alkylene-NH; Q is isopropyl; R_(1′″) is methoxy;and P comprises from 1 to 15 amino acid residues.
 50. The method ofclaim 49, wherein W is O; and P comprises 10 or fewer amino acidresidues.
 51. The method of claim 48, wherein P comprises from 1 toabout 20 amino acid residues.
 52. The method of claim 51, wherein Pcomprises an amino acid sequence which is a substrate for a matrixmetalloprotease.
 53. The method of claim 52, wherein the matrixmetalloprotease is selected from the group consisting of MMP-2, MMP-1,MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13 and MMP-26.
 54. The method ofclaim 53, wherein the matrix metalloprotease is MMP-2 or MMP-9.
 55. Themethod of claim 54, wherein P comprises the sequence -Pro-Leu-Gly-Xaa-,wherein Xaa is a naturally occurring amino acid residue.
 56. The methodof claim 55, wherein P comprises a sequence selected from the groupconsisting of Pro-Cha-Gly-Cys(Me)-His (SEQ ID NO:2);Pro-Gin-Gly-Ile-Ala-Gly-Gln-D-Arg (SEQ ID NO:3);Pro-Gln-Gly-Ile-Ala-Gly-Trp (SEQ ID NO:4);Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg (SEQ ID NO:5);Pro-Leu-Gly-Met-Trp-Ser-Arg (SEQ ID NO:35);Pro-Leu-Gly-Leu-Trp-Ala-D-Arg (SEQ ID NO:6); Pro-Leu-Ala-Leu-Trp-Ala-Arg(SEQ ID NO:7); Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:8);Pro-Leu-Ala-Tyr-Trp-Ala-Arg (SEQ ID NO:9); Pro-Tyr-Ala-Tyr-Trp-Met-Arg(SEQ ID NO:10); Pro-Cha-Gly-Nva-His-Ala (SEQ ID NO:11); Pro-Leu-Ala-Nva(SEQ ID NO:12); Pro-Leu-Gly-Leu (SEQ ID NO:13); Pro-Leu-Gly-Ala (SEQ IDNO:14); Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (SEQ ID NO:15);Pro-Cha-Ala-Abu-Cys(Me)-His-Ala (SEQ ID NO:16);Pro-Cha-Ala-Gly-Cys(Me)-His-Ala (SEQ ID NO:17);Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu (SEQ ID NO:18);Pro-Lys-Pro-Leu-Ala-Leu (SEQ ID NO:19);Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met (SEQ ID NO:20);Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:21);Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:22); andArg-Pro-Lys-Pro-Leu-Ala-Nva-Trp (SEQ ID NO:23).
 57. A method of treatinga subject having melanoma, comprising administering to said subject aneffective amount of a compound selected from the group consisting of{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3-methyl-butyric acid methyl ester;2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3-methyl-butyric acid methyl ester;2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-4-methyl-pentanoicacid methyl ester; {(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R, 3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-phenyl-aceticacid methyl ester; (1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S,5S, 6R )-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; (1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; (1-Hydroxymethyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; 2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3,3-dimethyl-butyricacid methyl ester; Cyclohexyl-2-{(3R, 4S, 5S, 6R )-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-aceticacid methyl ester; 2-{(3R, 4S, 5S, 6R )-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3-methyl-pentanoicacid methyl ester;[1-(1-Carbamoyl-2-hydroxy-ethylcarbamoyl)-2-methyl-propyl]-carbamicacid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R,3R)-2-methyl-3(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; 2-(3-{(3R, 4S, 5S, 6R )-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl}-ureido)-3-methyl-butyramide;2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonylamino}-3-methyl-butyricacid; N-Carbamoyl-Gly-Arg-Gly-Asp-Ser-Pro-(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester (SEQ ID NO:31); N-Carbamoyl-Gly-Arg-Gly-Asp-Tyr-(OMe)-Arg-Glu-(3R,4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester (SEQ ID NO:30); N-Carbamoyl-Gly-Arg-Gly-Asp-(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester (SEQ ID NO:32);N-Carbamoyl-Gly-Arg-Gly-3-amino-3-pyridyl-propionic acid-(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester (SEQ ID NO:40); N-Carbamoyl-Gly-Pro-Leu-Gly-Met-Trp-Ala-Gly-(3R,4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester (SEQ ID NO:39); N-Carbamoyl-Gly-Pro-Leu-(Me)Gly-(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl ester (SEQ ID NO:26); N-Carbamoyl-Gly-Pro-Leu-Gly-(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester (SEQ ID NO:27); Ac-Pro-Leu-Gly-Met-Trp-Ala-(2R-{(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol)ester (SEQ ID NO:24); Ac-Pro-Leu-Gly-Met-Gly-(2R-{(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol)ester (SEQ ID NO:36); Met-Trp-Ala-(2R-{(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol)ester (SEQ ID NO:37); Met-Gly-(2R-{(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol)ester (SEQ ID NO:38); Ac-Pro-Leu-Gly-Met-Ala-(2R-{(3R, 4S, 5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol) ester (SEQ ID NO:34);{2-Methyl-1-[methyl-(1-methyl-piperidin-4-yl)-carbamoyl]-propyl}-carbamicacid5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; [1-(2-Dimethylamino-ethylcarbamoyl)-2-methyl-propyl]-carbamicacid5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester;{1-[(2-Dimethylamino-ethyl)-methyl-carbamoyl]-2-methyl-propyl}-carbamicacid5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; [1-(3-Dimethylamino-propylcarbamoyl)-2-methyl-propyl]-carbamicacid5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester;[1-(3-Dimethylamino-2,2-dimethyl-propylcarbamoyl)-2-methyl-propyl]-carbamicacid5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; [2-Methyl-1-(4-methyl-piperazine-1-carbonyl)-propyl]-carbamicacid5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester;{2-Methyl-1-[2-(1-methyl-pyrrolidin-2-yl)-ethylcarbamoyl]-propyl}-carbamicacid 5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester;[2-Methyl-1-(4-pyrrolidin-1-yl-piperidine-1-carbonyl)-propyl]-carbamicacid5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; and [1-(4-Benzyl-piperazine-1-carbonyl)-2-methyl-propyl]-carbamicacid5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester; and pharmaceutically acceptable salts thereof.
 58. The method ofclaim 1, wherein said compound is administered to the subject using apharmaceutically acceptable formulation.
 59. The method of claim 20,wherein said compound is administered to the subject using apharmaceutically acceptable formulation.
 60. The method of claim 48,wherein said compound is administered to the subject using apharmaceutically acceptable formulation.
 61. The method of claim 57,wherein said compound is administered to the subject using apharmaceutically acceptable formulation.
 62. The method of claim 1,wherein said compound is administered to the subject intravenously,intramuscularly or orally.
 63. The method of claim 20, wherein saidcompound is administered to the subject intravenously, intramuscularlyor orally.
 64. The method of claim 48, wherein said compound isadministered to the subject intravenously, intramuscularly or orally.65. The method of claim 57, wherein said compound is administered to thesubject intravenously, intramuscularly or orally.
 66. The method ofclaim 1, wherein said subject is human.
 67. The method of claim 20,wherein said subject is human.
 68. The method of claim 48, wherein saidsubject is human.
 69. The method of claim 57, wherein said subject ishuman.
 70. The method of claim 1, wherein said compound is

or pharmaceutically acceptable salts thereof.
 71. A method of treating asubject having melanoma, comprising administering to said subject aneffective amount of a compound comprising the structure of

or pharmaceutically acceptable salts thereof.